“Pocket-sized RNA-Seq”: A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest

Currently, the discovery of new small ncRNAs requires high throughput methods even in the case of focused research on the regulation of specific genes or set of genes. We propose herein a simple, rapid, efficient, and cost effective method to clone and sequence single, yet unknown, small ncRNA. This technique that we called “Pocket-sized RNA-Seq” or psRNA-seq is based on in vitro transcription, RNA pull down and adapted RACE-PCR methods that allow its implementation using either available commercial kits or in-house reagents.

1. Produce bait RNAs following the MessageMuter™ shRNA production kit protocol. The final RNA pellet was resuspended in 20 µL of the 5X annealing buffer (provided in the kit) and 80 uL of RNase-free water.
Hints: The region of interest used as a bait can also be cloned in a vector under the control of a T7 promoter, PCR amplified or directly synthesized including the T7 promoter sequence.
Hints: Shorter is better; if the region producer of the miRNA is not known, it is better to use several overlapping shorter RNAs as baits than a longer one which will present highly structured RNA domains and will be more difficult to denature. 2. Concentration was first determined by A260 using NanoDrop spectrophotometer and further evaluated for appropriate size and absence of degradation by polyacrylamide gel (protocol from the MessageMuter™ shRNA production kit). 3. Mix 1 U CIP with 1 µg bait RNA in a 20 µL final volume. 4. Incubate at 37 °C for 1h. 5. RNAs were purified by phenol-chloroform extraction (see PHENOL-CHLOROFORM EXTRACTION section). 1. RNA samples were isolated from PBS-washed 10 6 cells using TRI reagent accordingly to manufacturer's instructions. RNA pellet was resuspended in 50 µL of RNase-free water. 2. Concentration was determined by A260. 3. Small RNAs were then size-fractionated using mirVana™ miRNA Isolation Kit accordingly to manufacturer's instructions and further evaluated for appropriate size and absence of degradation by polyacrylamide gel (protocol from the MessageMuter™ shRNA production kit).

Materials
Hints: The mirVana™ miRNA Isolation Kit proposed isolation of small RNA from cell lines using one unique protocol. However, we observed that small RNAs purified from total RNAs (themselves formerly extracted using Trizol or TRI reagent) were cleaner in a two-step procedure. Hints: Because of the small volume, denaturation using a thermal cycler with a heated lid would reduce evaporation.

Materials
2. Add 500 pmol of a specific biotinylated oligonucleotide (in 10 µL RNase-free water) and complete at 250 µL with RBB1.
Hints: For some RNAs, it might be more efficient (less background) to first hybridize biotinylated oligonucleotides and streptavidin magnetic beads, then to add in vitro transcribed RNA. 2. Polyadenylated captured small RNAs were purified by phenol-chloroform extraction (see PHENOL-CHLOROFORM EXTRACTION section). Hints: You can use your own RNA adapters. Moreover, a DNA adaptor can be used at 5′-end but the reverse transcription will also be less efficient.

Materials
3. Ligation products were purified by phenol-chloroform extraction (see PHENOL-CHLOROFORM EXTRACTION section). 4. Add 1 µL of GeneRacer oligo dT primer (50 µM) and 1 µL of dNTP (10 mM each) mix (provided in the kit) and incubate at 65 °C for 5 min. 5. Place on ice immediately to chill for 2 min. 6. Add 4 µL of 5X RT buffer, 15 U of AMV RT, 40 U of RNaseOUT and 2 µL of RNase-free water (all provided in GeneRacer kit) and incubate at 45 °C for 15 min. 7. Incubate at 85 °C for 15 min and spin briefly. 8. Amplify 1/100th of RT-PCR products by PCR using GeneRacer 5′ and 3′ primers (provided in the kit) using manufacturer's thermocycling and run 10-20 µL of the PCR product on 1% agarose gel + ethidium bromide.
Hints: It is sometimes useful to perform an additional nested PCR using internal primers.
9. Clone PCR products in pCR4-TOPO vector provided in the GeneRacer kit and sent for sequencing several clones.
Non-Coding RNA 2015, 1, 127-138; doi:10.3390/ncrna1020127 S6 2. Add 100 µL phenol:chloroform:isoamyl alcohol 25:24:1 and vortex vigorously for 30 s. 3. Centrifuge at maximum speed for 5 min and transfer aqueous phase (top) to a new tube. 4. Add 1 µL glycogen, 10 µL 3 M sodium acetate pH 5.5, 220 µL 100% ethanol and vortex briefly. 5. Place on ice for 10-20 min. 6. Centrifuge at maximum speed for 30 min at +4 °C and remove the supernatant carefully. 7. Wash with 500 µL 70% ethanol and centrifuge at maximum speed for 2 min at +4 °C. 8. Carefully remove the ethanol by pipet and air-dry the pellet for no more than 1-2 min at room temperature. 9. Resuspend the pellet in the appropriate volume of RNase-free water according to the step of protocol.

S7. Reagents and Solutions
Use deionized, distilled water in all recipes.  Figure S1. (A) Stem loop RT-PCR was used to evaluate the presence of a control miRNA (hsa-mir10a) and of the miRNA processed from hsa-primiR-21 (miR-21). Mature miRNAspecific primers were designed using miRNA Primer Design Tool Website and normalization was performed relative to the signal of RNU6-1 amplification (U6 RNA). (B) Ct obtained by stem-loop RT-qPCR using RNAs purified (short and long RNAs, < and > 200 nt, respectively, and total RNA) from MCF-7 and primary myoblast cells, and using stem-loop or random hexamer primers, with or without reverse transcriptase. Results are the mean ± standard deviation of at least 3 experiments per conditions performed in duplicates.
-5p and -3p, guide and star mature miR-21 strand, respectively; >, Ct were > 35 cycles; +RT and -RT, with or without reverse transcriptase, respectively. (C) Agarose gel of the PCR amplification of the final product. PCR products from reverse transcribed RNAs isolated and modified as described in the material and methods section, using 3 different baits related to miR-21 sequence (1-3) and one corresponding to the luciferase sequence. Left panel, psRNA-seq performed using MCF-7 cell line; right panel, psRNA-seq performed using primary cells (human myoblasts).