Studying miRNA–mRNA Interactions: An Optimized CLIP-Protocol for Endogenous Ago2-Protein

Transcriptome-wide analysis of RNA-binding partners is commonly achieved using UV crosslinking and immunoprecipitation (CLIP). Individual-nucleotide-resolution CLIP (iCLIP)enables identification of the specific position of the protein–RNA interaction. In addition to RNA-binding proteins (RBPs), microRNA (miRNA)–mRNA interactions also play a crucial role in the regulation of gene expression. Argonaute-2 (Ago2) mediates miRNA binding to a multitude of mRNA target sites, enabling the identification of miRNA–mRNA interactions by employing modified Ago2-CLIP protocols. Here, we describe an Ago2-specific CLIP protocol optimized for the use of small quantities of cell material, targeting endogenous Ago2 while avoiding possible methodological biases such as metabolic labeling or Ago2 overexpression and applying the latest advances in CLIP library preparation, the iCLIP2 protocol. In particular, we focus on the optimization of lysis conditions and improved radioactive labeling of the 5′ end of the miRNA.


Introduction
UV crosslinking and immunoprecipitation (CLIP) is a broadly used method to analyze interactions of RNA-binding proteins on a transcriptome-wide scale [1,2]. Via UV-A or UV-C photocrosslinking-the central feature of CLIP-covalent bonds between RNA and their protein interaction partners are formed; through protein-specific precipitation, these interactions can be analyzed on a transcriptome-wide level [3]. Several improvements and variations of the original CLIP protocol have been described since the first CLIP publication [4]; for example, [5] provides a comprehensive review and comparison of the most important CLIP variations. Broadly used adaptions allowing for optimized workflow for various purposes include enhanced CLIP (eCLIP) [6]; crosslinking and sequencing of hybrids (CLASH), which is highly valuable for host RNA-viral RNA interaction analyses [7]; high-throughput sequencing CLIP (HITS-CLIP) [8]; and covalent ligation of endogenous Argonaute-bound RNAs (CLEAR-CLIP) [9]. Additionally, individual-nucleotide-resolution CLIP (iCLIP) further allows for accurate binding site mapping by the terminating reverse transcription at the site of crosslinking [1,10]. Photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) involves the incorporation of photoreactive ribonucleoside analogs such as 4-thiouridine (4-SU) in order to increase the crosslinking efficiency of proteins to mRNA targets [11][12][13]. Several publications have shown alternatives to radioactive labeling of crosslinked and immunoprecipitated RNAs, such as PAR-CLIP with direct ligation of fluorescently labeled adapters to crosslinked RNA [14].
Argonaute 2 (Ago2), a component of the RNA-induced silencing complex (RISC), incorporates a multitude microRNAs (miRNA) as guides, each targeting a diverse set of messenger RNA (mRNA). By partially complementarily binding to mRNA, these miR-NAs downregulate translation and destabilize the mRNA, significantly impacting posttranscriptional regulation. In the case of completely complementary binding sites, mRNA cleavage and subsequent degradation are initiated, a mechanism often exploited when using small interfering RNAs (siRNA) [15]. In contrast to these inhibiting regulatory functions, miRNA have also been reported to bind to viral RNAs, to enhance translation and replication, and to stabilize the viral RNA genome in hepatitis C virus [16]. Thus, Ago2 is a valuable mediator for the analysis of miRNA-mRNA interactions. The first Ago2-CLIP was conducted with the PAR-CLIP method [17]. Subsequently, Ago2 has been employed, for example, in CLEAR-CLIP [9] and CLASH [7], protocols that allow for interaction analyses of miRNA with cellular mRNAs or viral RNAs [18].
Drawbacks of current Ago2-CLIP protocols range from the large quantities of required cellular starting material to the common use of overexpressed exogenous Ago2 or epitopetagged endogenous Ago2 in stable cell lines [11,14,17,19,20]. Moore et al. and several other groups have worked with endogenous Ago2 in their studies, mostly using brain tissue of different types of knockout mice or Halo-tagged Ago2 knock-in mice [8,9,20]. As brain tissue consists of several cell types, this may positively affect CLIP efficiency. Few CLIP experiments involving endogenous Ago2 have been described for human cell lines to date. These studies are limited to PAR-CLIP and cells with knockdown of RISC-related proteins potentially influencing Ago2 levels [2,11,18,[21][22][23]. Many of these experiments have been further described to require two to five 15 cm dishes per condition and replicate [24]. Kishore et al. additionally described an approach employing an additional micrococcal nuclease (MNase) treatment step to break down large RNA-protein complexes [2,17]. PAR-CLIP further introduces another potential experimental bias by adding 4-SU. This treatment allows for reduced crosslinking energy (365 nm UV-A light instead of 254 nm UV-C) and shorter exposure times, which enables recovery of a higher proportion of miRNA seed-complementary sites [2,25]. Toxicity due to a photoreactive nucleoside has not been observed in PAR-CLIP experiments [12]. However, changes in rRNA levels and reduced cell proliferation upon extended incubation have been reported, which may introduce biases in subsequent analyses [26].
Whereas the precipitation of RNA crosslinked to endogenous Ago2 is more challenging, it eliminates biases provoked by Ago2 overexpression or 4-SU addition, thus providing a more authentic snapshot of cellular miRNA-mRNA interactions [27]. This authenticity is of particular relevance for subsequent studies, for example, with respect to the interaction of miRNA with viral transcriptomes [16,18,28,29].
Based on the iCLIP2 protocol described by Buchbender et al. (2020) [1], we focused on overcoming the previously described biases and limitations by reducing the required cellular material and employing immunoprecipitation of untagged, endogenous Ago2 without addition of photoreactive nucleotides. The most important adjustments of this protocol include (i) the reduction in cellular starting materials, (ii) modification of lysis conditions, (iii) improved radioactive labeling of the miRNA 5 end, and (iv) immunoprecipitation in a cell-line-dependent manner. For new model systems, CLIP-related protocols have to be adjusted and optimized for the respective cell line or tissue. Here, we describe an optimized workflow of the iCLIP2-protocol and highlight the strategically most appropriate adaptations to this methodology.

Experimental Design
The following instructions are a combination of an analytical approach and a preparative approach to an optimized iCLIP2 protocol employing Ago2 (Figure 1). The analytical approach allows first insights into RNA-binding abilities of a protein or-in the case of Ago2, where RNA-binding ability is established-serves as a time-efficient approach to optimizing CLIP steps. The protocol can be shortened significantly using the analytical approach, as linker-ligations, RNA isolation, and sequencing are omitted. After establishing the optimized protocol, the preparative approach can be employed for RNA isolation and sequencing. and sequencing.
We found that the Ago2-iCLIP protocol, in addition to relying on the quality of the Ago2 antibody of choice, is also heavily dependent on the cell lines used. We used the Calu3 and an A549 cell lines (both human lung adenocarcinoma-derived). We suggest beginning with the protocol as described below, and if necessary, start by adjusting the lysis duration, salt concentrations of the stringent buffer, and RNase concentrations.  We found that the Ago2-iCLIP protocol, in addition to relying on the quality of the Ago2 antibody of choice, is also heavily dependent on the cell lines used. We used the Calu3 and an A549 cell lines (both human lung adenocarcinoma-derived). We suggest beginning with the protocol as described below, and if necessary, start by adjusting the lysis duration, salt concentrations of the stringent buffer, and RNase concentrations.  Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; Ensure the addition of protease inhibitor cocktail immediately before use. Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.

Procedure
The procedure is described for an analytical, as well as a preparative approach, as visualized in the experimental flow chart presented in Figure 1.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
For a general setup of the experiment, make sure to plan several controls appropriately, as shown in Figure 2. Proper labeling minimizes mix-up of samples and simplifies tracking of the samples and treatments.

5, x FOR PEER REVIEW 8 of 25
For a general setup of the experiment, make sure to plan several controls appropriately, as shown in Figure 2. Proper labeling minimizes mix-up of samples and simplifies tracking of the samples and treatments. First steps in setting up a CLIP experiment should include preliminary experiments, which are further described in the following protocol. The most prudent adjustments are shown in Figure 3 below.  First steps in setting up a CLIP experiment should include preliminary experiments, which are further described in the following protocol. The most prudent adjustments are shown in Figure 3 below. and low-RNase I concentrations, and negative controls First steps in setting up a CLIP experiment s which are further described in the following prot shown in Figure 3 below.  Beginning with Section 3.1. Bead preparation reduces the overall incubation time. During bead antibody incubation, lysate preparation (Section 3.2.) can be conducted, followed by DNase and partial RNase digestion (Section 3.3). For α-Ago2 clone 11A9, use Protein G Dynabeads. If another antibody is used, choose Protein A or G Dynabeads in an antibody-specific manner. Use the same Dynabeads for negative controls (e.g., α-FLAG or α-IgG antibody).

Bead Preparation for Immunoprecipitation; Time
to interactions without additional ligation steps [9,18,34]. RNA of the same sample different RNase concentrations can be pooled and labeled with the same barcode in sequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol develo by Buchbender et al. [1]. All essential steps are described below, but for more det explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per ple; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 12 RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at r temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sam on a magnet; Note: For an analytical approach, up to 50% of the volumes suffice, and recommended volumes differ depending on the approach.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are be disregarded here owing to the previous linker li Additionally, linker ligation has been reported to m tion partners to Ago2-incorporated miRNA, provid to interactions without additional ligation steps [9 different RNase concentrations can be pooled and sequent steps.
1. Prepare RNA-primer mix ( Table 7):  Note: The smaller volumes listed below have been shown to suffice, but increasing the α-Ago2 antibody amounts may increase the pulldown efficiency for some cell lines.
OPTIONAL ADJUSTMENT An antibody sandwich method can be used by incubating beads with~2 µg unconjugated α-IgG (rat) antibody for 1 h, washing 2× with cold TBS-T, and subsequently incubating with~0.5-1 µg α-Ago2 antibody for 1 h according to an analytical approach (see Section 5.3) [27]. This may increase Ago2-IP efficiency in some systems.

3.
Incubate on a wheel for 2 h at 4 • C. Continue with Sections 3.2 and 3.3. The CLIP protocol was optimized in the Calu3 and A549-ACE2 cell lines (both human lung adenocarcinoma-derived) cultured in DMEM with 10% FCS at 37 • C supplemented with 5% CO 2 for up to 35 passages.

Lysate Preparation
One or several 10-15 cm dishes of cells can be used for one CLIP experiment. Usually, one 15 cm dish of 70% confluence is sufficient for two to six subsequent samples (depending on the volume used for IP). OPTIONAL STEP Adding 100 µM 4-thiouridine (4-SU) to the cells 14 h before harvest increases the efficiency of Ago2-CLIP, as shown in several previous studies, but may introduce additional biases for subsequent analyses [12,26]

. MyONE Cleanup
Note: This protocol is designed for adherent cell lines. Thus, a crosslinking procedure needs to be adapted for non-adherent cells.

Crosslinking of Cells
Cells are crosslinked to covalently bind RNA to proteins in direct contact, providing a snapshot of RNA-protein interactions within living cells [8,27]. We suggest harvesting cells of 70 to 80% confluence. Excessively dense growth has been observed to decrease crosslinking efficiency.
Remove all liquid before crosslinking; 4.
Place culture dish in a tray with ice water for cooling and remove lid to crosslink cells under UV light according to Table 1

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

5.
Add 1 mL 1× PBS to cells, scrape to harvest, and collect in tube.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; CRITICAL STEP: Keep cells on ice unless stated otherwise; 6.
Remove all liquid.

First ProNex Size Selection
This step is conducted to remove primer-dimers.
1. Equilibrate ProNex chemistry to room temperature for 30 min; 2. Resuspend beads by vortexing vigorously; 3. For a 50 µL sample (PCR product), add 147.5 µL beads (1:2.95 v/v ratio of sample to beads); 4. Mix by pipetting 10 times up and down; 5. Incubate samples at room temperature for 10 min; 6. Place samples on a magnetic stand for 2 min, and discard the supernatant; Leave beads on the magnetic stand and add 300 µL ProNex wash buffer to the samples; incubate for 30-60 s before removal; Note: Ensure the wash buffer covers all beads on the magnet; CRITICAL STEP: Do not remove ProNex beads from magnet or resuspend during the wash; this can cause up to 20% sample loss. For large samples, increase the volume of ProNex wash buffer proportionally to the volume of samples and beads [1]; 7. Repeat the wash and allow sample to air dry for 8-10 min (<60 min) until cracking starts; 8. Remove the beads from the magnetic stand and start eluting the samples. Resuspend beads of the samples in 23 µL ProNex elution buffer; 9. Resuspend all samples by pipetting; then, let them incubate for 5 min at room temperature; 10. Return samples to the magnetic rack for 1 min; then, carefully transfer eluted cDNA to a clean tube.
PAUSE STEP: Freeze at −20 °C until further processing.
PAUSE STEP: Continue directly with Section 3.2.3; otherwise, snap freeze in liquid nitrogen and store at −80 • C until further use.

Cell Lysis
To disrupt as many unwanted protein-protein and protein-RNA interactions as possible, a stringent buffer containing ionic detergents is commonly used for cell lysis [3]. Here, we employed lysis buffer (non-ionic detergent) and RIPA buffer (ionic and non-ionic detergents). In accordance with Figure 3, preliminary testing should be conducted for the cell line and protein of interest.

Add 4 packed cell volumes (mg of pellet) of lysis buffer.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 17 of 25

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interac-tion partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in sub-sequent steps.
1. Prepare RNA-primer mix ( Table 7): 4. Add to sample (total volume 20 µL) and place in thermocycler at 25 °C for 5 min, 42 °C for 20 min, and 50 °C for 40 min, and hold at 4 °C; 5. Add 1.65 µL 1 M NaOH and incubate at 98 °C for 20 min to degrade the RNA and remove residual radioactive phosphates from the mixture in the subsequent cleanup step; 6. Add 20 µL 1 M HEPES-NaOH, pH 7.3; PAUSE STEP: Store cDNA at −20 °C until further use.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sam-ple; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; Note: When interested in the cytosolic Ago2 fraction, the use of lysis buffer is generally a favorable approach (Figure 4). RIPA buffer may improve the lysis of some cell lines and should be used for cases in which nuclear extracts are relevant. The required lysis conditions may differ depending on the cell type. Lysis buffer modifications include:

•
The addition of non-ionic SDS detergent (as described for RIPA buffer), which may improve lysis, in particular that of the nuclear fraction). The Addition of 0.5% sodium deoxycholate is a common approach to further dissociate and solubilize membrane-associated proteins [1,30]. For nuclear extracts, the addition of 1% Triton X-100 may be considered, which strongly permeabilizes the cell membrane and thus improves protein release [14]; • The addition of 0.5-1 mM ß-mercaptoethanol or dithiothreitol (DTT), which may inhibit the formation of large complexes and therefore improve immunoprecipitation [14,31]

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interac-tion partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in sub-sequent steps.
1. Prepare RNA-primer mix ( Table 7): 4. Add to sample (total volume 20 µL) and place in thermocycler at 25 °C for 5 min, 42 Note: This is generally used for bacterial and plant cells but may support the lysis of more resilient cells. The lysis efficiency should be tested in advance. For these first experiments, pellets and non-bound fractions should be included ( Figure 4). If the Ago2 Western blot signal is missing or low in the supernatant after lysis and most Ago2-protein is in the pellet after centrifugation of cell debris, sonication should be considered.

•
Depending on the sonicator, at least 1 mL of suspension may be required for sonication; thus, the sample size should be adjusted using additional input material or by increasing the lysis buffer volume per weight; • If the pellet is resuspended in RIPA buffer, the addition of detergents is suggested only after sonication to reduce foam formation.
Note: Ensure that the lysate volume (e.g., between tributed equally between all samples. The sample volu 100 and 250 µL for the analytical approach and at lea CLIP approach.
• Take 5-10% input material for Western control an 10× reducing agent, and water. Store at −20 °C unt

3.
Incubate on ice for 10 min and vortex vigorously every 3 min.
• OPTIONAL ADJUSTMENT: Extension of lysis incubation time from 10 min to ≥30 min on ice and vortexing every 10 min may improve protein concentrations for some cell lines (data not shown) [32].

4.
Centrifuge for ≥20 min at a minimum of 13,000 rpm at 4 • C.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7): Note: Ensure that the lysate volume (e.g., between low and high RNase) is distributed equally between all samples. The sample volume for IP should be between 100 and 250 µL for the analytical approach and at least 200 µL for the preparative CLIP approach.

•
Take 5-10% input material for Western control and add 4× LDS-loading buffer, 10× reducing agent, and water. Store at −20 • C until further use.

DNase and Partial RNase Digestion; Time for Completion: 20 min
Predetermine the appropriate RNase I concentrations according to an analytical approach. This should be done after adjustment of appropriate salt concentrations as described in Section 3.4.2. (Figure 3). We suggest a test experiment with a dilution series of RNase concentrations as provided in Table 2; expected results are documented in Section 4. Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol. Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; Note: For Ago2-CLIP with α-Ago2 Clone 11A9 in Calu3 and A549-ACE2 cells, we achieved satisfactory results with high (10 −3 ) to low (10 −5 ) dilutions.

4.
Add 1:100 volume of RNase I dilution to each sample and incubate for 3 min at 37 • C, shaking at 800 rpm. Fill the wells of thermomixer with water for more efficient heat conductivity; 5.
Immediately put on ice for ≥3 min.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7): Note: TBS800-T was found to be most efficient for stringent washing in these experiments, but the salt concentration can be verified by an IP test or by an analytical approach by comparison with, for example, TBS-600/800/1000-T (600/800/1000 mM NaCl) (Figure 3).
Add extract to prepared beads and rotate at 4 • C for 2 h; 3.
OPTIONAL STEP: Transfer the non-bound fraction to a new tube for Western blot controls and store at −20 • C until further use; 4.
Wash 4 times at 4 • C using TBS800-T; during the 3rd wash, transfer beads to fresh tubes. This prevents the elution of complexes unspecifically bound to the plastic of the tube when incubating with sample loading buffer. This may reduce the background when no SafeSeal reaction tubes are used; Ago2 was previously suggested to fold around the 5 end of the incorporated miRNA, thus reducing accessibility for the T4 polynucleotide kinase (T4 PNK) and incorporation of [gamma-32P]-ATP [27]. We found that 5 end dephosphorylation using recombinant shrimp alkaline phosphatase (rSAP) prior to labeling improved radioactive labeling in this approach, in particular for Ago2-miRNA-mRNA complexes (red arrow in Figure 5).
Total volume: 40.00 2. Add 40 µL phosphatase mix to beads and incubate at 37 °C 3. Wash 2 times with TBS400-T and transfer to fresh tube duri 4. Wash 2 times with PNK wash buffer.
PAUSE STEP: Analytical approach: Store beads in PNK 4 °C or continue with Section 3.6. Figure 5. Analytical approach of Ago2-iCLIP2. Radiography of expec using protein marker VI (pre-stained) peqGOLD. A volume of 10 µL of was run for 90 min at 200 V, and transferred to an Amersham Protran membrane for 1 h at 30 V. The sample was subsequently exposed to plate overnight. Calu3 cells were crosslinked at 254 nm with 450 mJ/cm 4× packed cell volume lysis buffer. IP with 0.5 µg α-Ago2 clone 11A9 dilutions of 10 −3 and 10 −5 , and with or without 5′ end dephosphorylation tive labeling with ATP mix (1/2 volume [gamma-32P]-ATP). The red bo est with an RNase I concentration-dependent Ago2-bound RNA sme Ago2-miRNA-mRNA complexes, which are more pronounced after 5′ e sequent Western blot analysis of protein levels of Ago2 on CLIP-membra 11A9 as primary antibody in 5% milk-TBS-T and 1:10,000 α-rat IgG-H Visualization on Amersham Hyperfilm ECL film. Figure 5. Analytical approach of Ago2-iCLIP2. Radiography of expected results on NuPAGE gel using protein marker VI (pre-stained) peqGOLD. A volume of 10 µL of sample was loaded, the gel was run for 90 min at 200 V, and transferred to an Amersham Protran 0.45 µm NC nitrocellulose membrane for 1 h at 30 V. The sample was subsequently exposed to a Fuji BAS-SR2040 imaging plate overnight. Calu3 cells were crosslinked at 254 nm with 450 mJ/cm 2 and lysed by sonication in 4× packed cell volume lysis buffer. IP with 0.5 µg α-Ago2 clone 11A9, RNase I digest employing dilutions of 10 −3 and 10 −5 , and with or without 5 end dephosphorylation using rSAP before radioactive labeling with ATP mix (1/2 volume [gamma-32P]-ATP). The red box indicates the area of interest with an RNase I concentration-dependent Ago2-bound RNA smear. The red arrow indicates Ago2-miRNA-mRNA complexes, which are more pronounced after 5 end dephosphorylation. Subsequent Western blot analysis of protein levels of Ago2 on CLIP-membrane, with 1:500 α-Ago2 clone 11A9 as primary antibody in 5% milk-TBS-T and 1:10,000 α-rat IgG-HRP as secondary antibody. Visualization on Amersham Hyperfilm ECL film.
Add 40 µL phosphatase mix to beads and incubate at 37 • C for 20 min; 3.
Wash 2 times with TBS400-T and transfer to fresh tube during 2nd wash; 4.
Wash 2 times with PNK wash buffer.
2. Run PCR (Table 11): PAUSE STEP: Analytical approach: Store beads in PNK wash buffer overnight at 4 • C or continue with Section 3.6.  Prepare phosphatase mix to dephosphorylate the 3 end. The low pH of PNK dephosphorylation buffer enables dephosphorylation by T4 PNK (Table 4);  to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; Prepare PNK mix ( Table 6):

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; Note: Our experience shows that Ago2-CLIP can effectively be conducted with a mix of 1 part [gamma-32P]-ATP and 1 part non-radioactive ATP (same concentrations) per sample. Ratios of [gamma-32P]-ATP and ATP can be adjusted further to reduce or increase the radioactive signal. Whereas a stronger signal reduces exposure times of X-ray films, additional radioactivity will be transferred into the RNA isolation.

2.
Add to beads and incubate in thermomixer at 37 • C for 20 min at 1000 rpm; 3.
Wash once with cold TBS-T; 4.
Wash once with cold PNK wash buffer; 5.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7): CRITICAL STEP: It is crucial to use a protein gel system with a neutral pH to prevent RNA degradation by alkaline hydrolysis [1]. The Invitrogen NuPAGE system meets these requirements. Alternatively, other gel systems that retain neutral pH during the electrophoresis run, e.g., as described by Graham et al., can be used [33].

1.
Use a NuPAGE system and 4-12% Bis-Tris gels in 1× NuPAGE MOPS SDS running buffer; • Analytical approach: gels with a well size of 1.0 mm are sufficient; • Preparative approach: use gels with a well size of 1.5 mm to load more volume and yield a higher RNA amount for sequencing.
Load inputs and samples in appropriate volume for well size; 4.
Run gel for a minimum of 2 h at 200 V.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; Note: The relevant area is above the band of the protein of interest (above 100 kDa); thus, the gel run can be extensive. For Ago2-CLIP, a 2 to 3 h gel run is optimal.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; If analyzing truncated constructs, the duration should be adjusted accordingly.

Transfer
Using the wet transfer chamber approach of the NuPAGE system, gel is transferred onto a nitrocellulose membrane. This is an additional cleanup step, as only RNAs covalently crosslinked to precipitated proteins are transferred. Free RNA does not bind to nitrocellulose with sufficient affinity.
Set up the system according to the manufacturer's protocol; 3.
Blot at 30 V for 1 h or at 15 V overnight; 4.
Draw marker bands with a radioactive pen onto a piece of plastic bag and place on/next to membrane; 5.
Place inside a plastic bag; 6.
Expose the membrane; •   Use X-ray film as a reference and cut the region of interest from the CLIP membrane ( Figure 6); 2.
Add 400 µL of PK-urea buffer and incubate for an additional 20 min at 55 • C while shaking; 5.
Separate phases by spinning for 5 min at 13,000 rpm at 4 • C using Phase Lock Gel Heavy 2 mL tubes; 8.
Transfer aqueous layer into a new tube without touching the wax; 9.
Precipitate by adding 1 µL GlycoBlue and 80 µL 3 M sodium acetate, pH 5.   PAUSE STEP: Store in 70% ethanol at −80 • C until further processing; 12. Dissolve pellets in 12 µL water; OPTIONAL STEP: As a control, perform a Western blot of the remaining nitrocellulose membrane under protective measures in an isotope laboratory.
PAUSE STEP: Store in 70% ethanol at −80 °C until further 12. Dissolve pellets in 12 µL water; OPTIONAL STEP: As a control, perform a Western blot of th lose membrane under protective measures in an isotope labo Then, the sample was exposed to Amersham Hyperfilm MP high-performance autoradiography film for five days. Blue boxes indicate regions of interest on the membrane, which was cut for further processing. Red arrows indicate Ago2-miRNA-mRNA and Ago2-miRNA complexes. Subsequent Western blot on CLIP-membrane. α-Ago2 clone 11A9 (1:500) in 5% milk-TBS-T, followed by α-rat IgG. Exposure with Lumi-Light Plus on Amersham Hyperfilm ECL film.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; CRITICAL STEP: Never put on ice; 3.

First ProNex Size Selection
This step is conducted to remove primer-dimers.
1. Equilibrate ProNex chemistry to room temperature for 30 min; 2. Resuspend beads by vortexing vigorously; 3. For a 50 µL sample (PCR product), add 147.5 µL beads (1:2.95 v/v ratio of sample to beads); 4. Mix by pipetting 10 times up and down; 5. Incubate samples at room temperature for 10 min; 6. Place samples on a magnetic stand for 2 min, and discard the supernatant; Leave beads on the magnetic stand and add 300 µL ProNex wash buffer to the samples; incubate for 30-60 s before removal; Note: Ensure the wash buffer covers all beads on the magnet; CRITICAL STEP: Do not remove ProNex beads from magnet or resuspend during the wash; this can cause up to 20% sample loss. For large samples, increase the volume of ProNex wash buffer proportionally to the volume of samples and beads [1]; 7. Repeat the wash and allow sample to air dry for 8-10 min (<60 min) until cracking starts; 8. Remove the beads from the magnetic stand and start eluting the samples. Resuspend beads of the samples in 23 µL ProNex elution buffer; 9. Resuspend all samples by pipetting; then, let them incubate for 5 min at room temperature; 10. Return samples to the magnetic rack for 1 min; then, carefully transfer eluted cDNA to a clean tube.
PAUSE STEP: Freeze at −20 °C until further processing.
PAUSE STEP: Store cDNA at −20 • C until further use.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3.
Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4.
Place beads on a magnetic rack and discard the supernatant; 5.
Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6.
Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample on a magnet; 7.
Spin down in a microcentrifuge, place on a magnet, and discard the supernatant; 8.
Air dry beads for 5 min at room temperature and resuspend in 5 µL water;

9.
Incubate the mix at room temperature for 5 min and Methods Protoc. 2022, 5, x FOR PEER REVIEW

Reverse Transcription
Whereas miRNAs with a length of 17be disregarded here owing to the previous Additionally, linker ligation has been repo tion partners to Ago2-incorporated miRNA to interactions without additional ligation different RNase concentrations can be poo sequent steps.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7):  Note: At this point, samples should be free of residual radioactive ATP and RNA fragments. After verifying this, the experiment can be continued outside an of isotope laboratory.

Second Linker Ligation to 3 End of cDNA
Choose different 3 cDNA linkers for each experiment and control (Table 9). Barcoding enables pooling of several experiments before sequencing. Using every possible base at every position or using A at every position of the experimental barcode facilitates 2-color Illumina sequencing [1]. Heat the mix for 2 min at 75 • C and immediately put on ice for >1 min; 3.
Prepare the ligation mix on ice. Mix by vigorously stirring, pipetting, and flicking to ensure homogeneity. Briefly spin down in a microcentrifuge; 4.
Add another 1 µL RNA ligase to each sample to a final volume of 21 µL and mix by stirring; 6.

First ProNex Size Selection
This step is conducted to remove primer-dimers.
1. Equilibrate ProNex chemistry to room temperature for 30 min; 2. Resuspend beads by vortexing vigorously; 3. For a 50 µL sample (PCR product), add 147.5 µL beads (1:2.95 v/v ratio of sample to beads); 4. Mix by pipetting 10 times up and down; 5. Incubate samples at room temperature for 10 min; 6. Place samples on a magnetic stand for 2 min, and discard the supernatant; Leave beads on the magnetic stand and add 300 µL ProNex wash buffer to the samples; incubate for 30-60 s before removal; Note: Ensure the wash buffer covers all beads on the magnet; CRITICAL STEP: Do not remove ProNex beads from magnet or resuspend during the wash; this can cause up to 20% sample loss. For large samples, increase the volume of ProNex wash buffer proportionally to the volume of samples and beads [1]; 7. Repeat the wash and allow sample to air dry for 8-10 min (<60 min) until cracking starts; 8. Remove the beads from the magnetic stand and start eluting the samples. Resuspend beads of the samples in 23 µL ProNex elution buffer; 9. Resuspend all samples by pipetting; then, let them incubate for 5 min at room temperature; 10. Return samples to the magnetic rack for 1 min; then, carefully transfer eluted cDNA to a clean tube.
PAUSE STEP: Freeze at −20 °C until further processing.
PAUSE STEP: Agitate overnight at room temperature and 1100 rpm.

MyONE Cleanup 2
Fresh MyONE beads are added to the cDNA-bead mix from the ligation reaction (Section 3.8.4).
Wash beads with 500 µL RLT buffer and resuspend them in 60 µL RLT buffer; add to the sample and mix; 3.
Add 60 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for another 5 min; 4.
Place beads on a magnetic rack and discard the supernatant; 5.
Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6.
Wash 2 times with 80% ethanol; incubate 30 s each time before putting the sample on the magnet; 7.
Spin in a microcentrifuge, place on the magnet, and discard supernatant; 8.
Air dry beads for 5 min at room temperature and resuspend in 23 µL water; 9.
Incubate the mix for 5 min at room temperature; 10. Magnetically attract beads and add eluate to PCR mix of 3.9. Prepare the PCR mix (Table 10):

2.
Run PCR (Table 11):  Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol. Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; 6. Wash 2 times with 80% ethanol. Incubate 30 sec each time before placing the sample CRITICAL STEP: Do not remove ProNex beads from magnet or resuspend during the wash; this can cause up to 20% sample loss. For large samples, increase the volume of ProNex wash buffer proportionally to the volume of samples and beads [1]; 7.
Repeat the wash and allow sample to air dry for 8-10 min (<60 min) until cracking starts; 8.
Remove the beads from the magnetic stand and start eluting the samples. Resuspend beads of the samples in 23 µL ProNex elution buffer; 9.
Resuspend all samples by pipetting; then, let them incubate for 5 min at room temperature; 10. Return samples to the magnetic rack for 1 min; then, carefully transfer eluted cDNA to a clean tube.  The PCR test should be performed with two or three different cycle numbers to determine the optimal cycles for the preparative PCR. For Ago2-CLIP, we recommend 14 to 20 cycles for the first PCR test.

1.
Prepare the PCR mix for two/three cycle numbers (

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix (Table 7): CRITICAL STEP: At this point, the samples should not be opened in the same working space as before. If possible, switch to a designated post-PCR bench or even room and use different pipettes and equipment. Using the same pipettes as before may contaminate future samples with PCR products of previous experiments, falsifying the sequencing results;
Use 0.5-1 µL of GeneRuler Low-Range DNA Ladder diluted in a total volume of 6 µL 1× loading buffer as a size marker, and load alongside samples;

5.
Run on 7% PAA-TBE gel for 30 min at 200 V and stain for 10 min with ethidium bromide solution.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7):   The second cDNA size selection eliminates excess primer that may negatively influence sequencing results. Here, a different sample-to-ProNex bead ratio than in the first size selection is required because primers in preparative PCR are longer in comparison to cDNA preamplification (Table 15). Whereas miRNAs with a length of 17-24 nt are difficult to re be disregarded here owing to the previous linker ligation, which Additionally, linker ligation has been reported to mediate the li tion partners to Ago2-incorporated miRNA, providing an additi to interactions without additional ligation steps [9,18,34]. RNA different RNase concentrations can be pooled and labeled with sequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iC by Buchbender et al. [1]. All essential steps are described below explanations of these parts, refer to the original protocol.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7):  The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
Note: Ensure that the wash buffer covers all beads on the magnet; incubate for 30-60 s before removal.
Remove the beads from the magnetic rack and start eluting the samples. Be sure to elute beads of the samples in 20 µL ProNex elution buffer; 10. Resuspend all samples by pipetting and let them stand for 5 min at room temperature; 11. Return the samples to the magnetic stand for 1 min; then, carefully transfer the eluted cDNA to a clean tube; 12. Pool half of each sample in one reaction tube; 13. Use 1% of library to assess primer removal on a 7% PAA-TBE gel; • Combine 1% library with water and 1 µL 6× DNA-loading buffer; 14. Analyze on 7% PAA-TBE gel together with previously pooled inputs (step 4) for 25 min to determine primers.
Half of the prepared library can be sent to sequencing facilities for bioanalyzer analyses and sequencing runs.

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.

•
For limited sample and linker numbers, spike in for sequencing is recommended.

DNase and Partial RNase Digestion
Preliminary testing of RNase I concentrations should be performed to determine the most effective concentrations, as described in Section 3.3. A smear is visible above the protein of interest in the radiography. With lower RNase I concentrations, the smear increases in size, owing to a lower degree of RNA degradation ( Figure 5, red box).

Immunoprecipitation
Immunoprecipitation should be tested beforehand to ensure antibody efficiency. Additionally, lysis conditions can be optimized. Figure 4 shows Western blots of preliminary IPs with lysis buffer in comparison with RIPA buffer and including all fractions of immunoprecipitation.

Radiography
It has been reported that Ago2-miRNA complexes show a radioactive signal at 110 kDa, whereas Ago2-miRNA-mRNA complexes show a signal at >130 kDa [8,27]. The radiography should show a smear, specifically in the area above the protein of interest. Smear intensity increases with lower RNase I concentrations, as less RNA has been degraded ( Figure 5).
In the subsequent preparative approach, the nitrocellulose membrane after transfer is exposed to an X-ray film. The X-ray film serves as a template to cut out the appropriate fragments. A Western blot is performed subsequently to analyze immunoprecipitation efficiency ( Figure 6).
After the second PCR amplification test run, cDNA is loaded on 7% PAA-TBE gels. Bands for miRNA should run at around 154 nt, as the total linker length is 134 nt, and miRNA length is~20 nt [1]. Bands of larger sizes indicate miRNA-mRNA hybrids, as previously visualized by Moore et al. [27].

Further Explorations and Troubleshooting
As previously mentioned, important adjustments include the salt concentration of stringent washing, cell-line-dependent crosslinking, lysis conditions, and RNase I dilutions ( Figure 3). These can be adjusted as described above. Below, we detail antibody restrictions and list further potential modifications.

α-Ago2 Antibody Choice
Choosing an α-Ago2 antibody is not a trivial task, considering the variety of available antibodies. In our experiments, we tested several antibodies with varying results. Precipitation with α-Ago2 antibody clone 9E8.2 (Merck KGaA, Darmstadt, Germany, Cat. no.: 04-642) was functionally limited, and detection was only possible on a PVDF membrane. Because iCLIP2 relies on transfer onto nitrocellulose, this antibody was not further employed. Using the Anti-pan Ago antibody clone 2A8 (Merck KGaA, Darmstadt, Germany, Cat. no.: MABE56) also showed no signals in preliminary IPs. Western blot of α-Ago2 clone 11A9 in 5% milk-TBS-T yielded an adequate protein signal and indicated successful IP; thus, all subsequent experiments were conducted with this antibody.

Depletion of Large Ribonucleoprotein Particles (RNPs)
A step commonly described in immunoprecipitation protocols (after Section 3.4.2 step 1) is the removal of large RNPs by centrifugation for 5 min at 16,400 rpm at 4 • C [1]. This has been shown to substantially reduce the RNA signal in the radiography in our experiments (data not shown). Thus, we advise excluding this step from the immunoprecipitation protocol.

Antibody Sandwich Method
Moore et al. first described a sandwich antibody method to increase immunoprecipitation efficiency in CLIP experiments [27]. Incubation of Dynabeads with α-IgG before incubation with α-Ago2 may reduce the effects of steric hindrance and increase the binding efficiency of α-Ago2 to the beads via the mediating antibody. Additionally, the intermediate offers extra binding sites for α-Ago2, as illustrated in Figure 7.
Moore et al. first described a sandwich anti itation efficiency in CLIP experiments [27]. Incu incubation with α-Ago2 may reduce the effects o ing efficiency of α-Ago2 to the beads via the me mediate offers extra binding sites for α-Ago2, as  [27], we suggest reducing the ratio of bridgi beginning with an analytical approach.
Note: Choose an appropriate α-IgG anti on the organism of origin (α-rat IgG for α-Ago2 Note: In our experiments, we did not ob method. In other cell types or when using differ approach may improve Ago2-IP efficiency [27].

Suggested Explorations
In case further optimization is necessary fo tions for improvement are applicable.
Labeling efficiency may be improved by mix of ATP [35]. Another option is radioactive 3′ end minal transferases, an approach which has bee Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size. Additionally, linker ligation has been reported to mediate the ligation of mRNA interaction partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in subsequent steps.
1. Prepare RNA-primer mix ( Table 7): 4. Add to sample (total volume 20 µL) and place in thermocycler at 25 °C for 5 min, 42 °C for 20 min, and 50 °C for 40 min, and hold at 4 °C; 5. Add 1.65 µL 1 M NaOH and incubate at 98 °C for 20 min to degrade the RNA and remove residual radioactive phosphates from the mixture in the subsequent cleanup step; 6. Add 20 µL 1 M HEPES-NaOH, pH 7.3; PAUSE STEP: Store cDNA at −20 °C until further use.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sample; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; 4. Place beads on a magnetic rack and discard the supernatant; 5. Resuspend beads in 1 mL 80% ethanol and transfer the mix to a new tube; Note: Choose an appropriate α-IgG antibody for the α-Ago2 antibody depending on the organism of origin (α-rat IgG for α-Ago2 clone 11A9).

Reverse Transcription
Whereas miRNAs with a length of 17-24 nt are difficult to reverse transcribe, this can be disregarded here owing to the previous linker ligation, which increases fragment size.
Additionally, linker ligation has been reported to mediate the ligation of mRNA interac-tion partners to Ago2-incorporated miRNA, providing an additional insight with respect to interactions without additional ligation steps [9,18,34]. RNA of the same sample and different RNase concentrations can be pooled and labeled with the same barcode in sub-sequent steps.

MyONE Cleanup
The following steps of the protocol are adopted from the iCLIP2 protocol developed by Buchbender et al. [1]. All essential steps are described below, but for more detailed explanations of these parts, refer to the original protocol.
1. Mix magnetic MyONE silane bead solution thoroughly and use 10 µL beads per sam-ple; 2. Wash beads with 500 µL RLT buffer using a magnetic stand and resuspend in 125 µL RLT buffer; add to sample and mix; 3. Add 150 µL 100% ethanol and carefully mix by pipetting; incubate for 5 min at room temperature, mix, and incubate for an additional 5 min; Note: In our experiments, we did not observe significant improvement using this method. In other cell types or when using different lysis methods, the bridging antibody approach may improve Ago2-IP efficiency [27].

Suggested Explorations
In case further optimization is necessary for the applied cell line, the following options for improvement are applicable.
Labeling efficiency may be improved by mixing [gamma-32P]-ATP with ADP instead of ATP [35]. Another option is radioactive 3 end labeling using [alpha-32P]-ATP and terminal transferases, an approach which has been reported to result in reduced labeling efficiency and thus longer autoradiography exposure times [27,36].
The use of radioactivity is not mandatory for CLIP procedures. Protocols have been published using direct ligation of a fluorescently labeled adapter to the 3' end of crosslinked RNA. Refer to [14] for a detailed protocol on non-radioactive PAR-CLIP for small RNA library preparations.