An Efficient Chromatin Immunoprecipitation Protocol for the Analysis of Histone Modification Distributions in the Brown Alga Ectocarpus

The brown algae are an important but understudied group of multicellular marine organisms. A number of genetic and genomic tools have been developed for the model brown alga Ectocarpus; this includes, most recently, chromatin immunoprecipitation methodology, which allows genome-wide detection and analysis of histone post-translational modifications. Post-translational modifications of histone molecules have been shown to play an important role in gene regulation in organisms from other major eukaryotic lineages, and this methodology will therefore be a very useful tool to investigate genome function in the brown algae. This article provides a detailed, step-by-step description of the Ectocarpus ChIP protocol, which effectively addresses the difficult problem of efficiently extracting chromatin from cells protected by a highly resistant cell wall. The protocol described here will be an essential tool for the future application of chromatin analysis methodologies in brown algal research.


Introduction
Brown algae (Phaeophyceae) are a group of almost exclusively marine photosynthetic eukaryotes. These seaweeds are found along coastlines worldwide, where they have important ecological roles as primary producers and as habitats for a broad range of other species [1]. In recent years, several brown algal species have emerged as important aquaculture crops as a consequence of their capacity to rapidly produce biomass under sustainable cultivation conditions that do not require the use of arable terrestrial land or freshwater resources [2,3]. All brown algae are multicellular, and some species, such as the kelps, exhibit considerable developmental complexity. As members of the Stramenopile supergroup, these seaweeds are very distantly related to other complex multicellular groups, such as animals, land plants, and fungi [4]. Brown algae therefore acquired complex multicellularity independent of their animal and land plant lineages and, consequently, are of interest in order to understand this important evolutionary transition [5].
The small, filamentous alga Ectocarpus has been established as a genetic model organism for the brown algae [4,6], and a range of genetic and genomic tools are available. These include the complete genome sequence of Ectocarpus strain Ec32, which is publicly available at http://bioinformatics.psb.ugent.be/orcae/overview/EctsiV2 (accessed on 21 Figure 1. Overview of the ChIP protocol. After harvesting, the Ectocarpus material is treated with formaldehyde to crosslink the protein and DNA components of the chromatin. Chromatin is then extracted by isolating and subsequently lysing nuclei. After fragmentation of the isolated chromatin, DNA fragments crosslinked to histone proteins bearing a post-translational modification of interest are isolated by immunoprecipitating with an antibody raised against the histone PTM. Following reverse crosslinking, the isolated DNA can be recovered and sequenced. Large-size cell strainer (for example, a 12.5 cm Finlandek permanent coffee filter) (optional, for tissue harvesting after culture)  Dissection forceps Figure 1. Overview of the ChIP protocol. After harvesting, the Ectocarpus material is treated with formaldehyde to crosslink the protein and DNA components of the chromatin. Chromatin is then extracted by isolating and subsequently lysing nuclei. After fragmentation of the isolated chromatin, DNA fragments crosslinked to histone proteins bearing a post-translational modification of interest are isolated by immunoprecipitating with an antibody raised against the histone PTM. Following reverse crosslinking, the isolated DNA can be recovered and sequenced.  [24]. Culture at least 1200 individual sporophytes or gametophytes at a density of six individuals per Petri dish. 2. After culture, carefully transfer the algae with dissection forceps into a sterile Erlenmeyer flask containing 400 mL of sterile seawater. Two weeks of culture should produce approximately 4-5 g (FW) of tissue.

Materials and Reagents
CRITICAL STEP For some strains it may be necessary to grow more individuals per Petri dish and harvest at an earlier stage of development before cultures become fertile and produce the next generation of the life cycle. With some sporophyte strains it may be necessary to use a cell scraper to dislodge thalli from the bottom of the Petri dish. This material can be collected by filtering the seawater medium and then transferring the algal material with dissection forceps to the 400 mL of sterile seawater.

Crosslinking (Required Time: 2 h)
3. Crosslink the algal material by adding 400 mL of 2x crosslinking buffer to the 400 mL of algal material in seawater and incubating for exactly 5 min at room temperature under a chemical hood. Mix gently during the crosslinking.
CRITICAL STEP Do not incubate for more than 5 min in the 2x crosslinking buffer as this could lead to excessive crosslinking, which may interfere with the chromatin extraction and immunoprecipitation steps. Note that formaldehyde is toxic if inhaled, ingested, or absorbed through skin. 4. Filter the tissue rapidly (15 s maximum) through a sterile piece of Miracloth to eliminate the formaldehyde. Transfer the tissue to a new 50 mL tube containing 50 mL of PBS quenching buffer for 5 min at room temperature. Mix gently during quenching. 5. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes or for 10 min for sporophytes. 6. Eliminate the buffer and resuspend in 50 mL of 1x phosphate-buffered saline (PBS) to wash the tissue. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes, or for 10 min for sporophytes. 7. Remove as much of the supernatant as possible by pipetting. You can invert the tube onto a piece of Miracloth placed on absorbent paper. Wrap approximately 1 g batches of tissue in aluminium foil, note the mass of tissue in each batch as this will be used to calculate the volume of nuclei isolation buffer to be added in the following steps. Freeze the crosslinked tissue rapidly in liquid nitrogen.

PAUSE STEP
The crosslinked tissue can be stored at −80 °C at this stage, but it should be used within the next few days if possible, and should not be stored for more than 1 month.
CRITICAL STEP The filamentous structure of the material may be partly dissociated after fixation, making it difficult to recover the pellet. If this is the case, scoop the pellet out of the Falcon tube with a spatula.

Isolation of Semipure Nuclei (Required Time: 2 h)
8. Grind about 1 g of crosslinked tissue to an ultra-fine powder under liquid nitrogen using a pre-chilled mortar and pestle. Ensure that samples do not thaw during grinding.
CRITICAL STEP For some strains it may be necessary to grow more individuals per Petri dish and harvest at an earlier stage of development before cultures become fertile and produce the next generation of the life cycle. With some sporophyte strains it may be necessary to use a cell scraper to dislodge thalli from the bottom of the Petri dish. This material can be collected by filtering the seawater medium and then transferring the algal material with dissection forceps to the 400 mL of sterile seawater.

3.
Crosslink the algal material by adding 400 mL of 2x crosslinking buffer to the 400 mL of algal material in seawater and incubating for exactly 5 min at room temperature under a chemical hood. Mix gently during the crosslinking.  [24]. Culture at least 1200 individual sporophytes or gametophytes at a density of six individuals per Petri dish. 2. After culture, carefully transfer the algae with dissection forceps into a sterile Erlenmeyer flask containing 400 mL of sterile seawater. Two weeks of culture should produce approximately 4-5 g (FW) of tissue.
CRITICAL STEP For some strains it may be necessary to grow more individuals per Petri dish and harvest at an earlier stage of development before cultures become fertile and produce the next generation of the life cycle. With some sporophyte strains it may be necessary to use a cell scraper to dislodge thalli from the bottom of the Petri dish. This material can be collected by filtering the seawater medium and then transferring the algal material with dissection forceps to the 400 mL of sterile seawater.

Crosslinking (Required Time: 2 h)
3. Crosslink the algal material by adding 400 mL of 2x crosslinking buffer to the 400 mL of algal material in seawater and incubating for exactly 5 min at room temperature under a chemical hood. Mix gently during the crosslinking.
CRITICAL STEP Do not incubate for more than 5 min in the 2x crosslinking buffer as this could lead to excessive crosslinking, which may interfere with the chromatin extraction and immunoprecipitation steps. Note that formaldehyde is toxic if inhaled, ingested, or absorbed through skin. 4. Filter the tissue rapidly (15 s maximum) through a sterile piece of Miracloth to eliminate the formaldehyde. Transfer the tissue to a new 50 mL tube containing 50 mL of PBS quenching buffer for 5 min at room temperature. Mix gently during quenching. 5. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes or for 10 min for sporophytes. 6. Eliminate the buffer and resuspend in 50 mL of 1x phosphate-buffered saline (PBS) to wash the tissue. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes, or for 10 min for sporophytes. 7. Remove as much of the supernatant as possible by pipetting. You can invert the tube onto a piece of Miracloth placed on absorbent paper. Wrap approximately 1 g batches of tissue in aluminium foil, note the mass of tissue in each batch as this will be used to calculate the volume of nuclei isolation buffer to be added in the following steps. Freeze the crosslinked tissue rapidly in liquid nitrogen.

PAUSE STEP
The crosslinked tissue can be stored at −80 °C at this stage, but it should be used within the next few days if possible, and should not be stored for more than 1 month.
CRITICAL STEP The filamentous structure of the material may be partly dissociated after fixation, making it difficult to recover the pellet. If this is the case, scoop the pellet out of the Falcon tube with a spatula.

Isolation of Semipure Nuclei (Required Time: 2 h)
8. Grind about 1 g of crosslinked tissue to an ultra-fine powder under liquid nitrogen using a pre-chilled mortar and pestle. Ensure that samples do not thaw during grinding.
CRITICAL STEP Do not incubate for more than 5 min in the 2x crosslinking buffer as this could lead to excessive crosslinking, which may interfere with the chromatin extraction and immunoprecipitation steps. Note that formaldehyde is toxic if inhaled, ingested, or absorbed through skin.

4.
Filter the tissue rapidly (15 s maximum) through a sterile piece of Miracloth to eliminate the formaldehyde. Transfer the tissue to a new 50 mL tube containing 50 mL of PBS quenching buffer for 5 min at room temperature. Mix gently during quenching.
Eliminate the buffer and resuspend in 50 mL of 1x phosphate-buffered saline (PBS) to wash the tissue. Centrifuge at 3215× g 4 • C in an Eppendorf 5804R for 5 min for gametophytes, or for 10 min for sporophytes. 7.
Remove as much of the supernatant as possible by pipetting. You can invert the tube onto a piece of Miracloth placed on absorbent paper. Wrap approximately 1 g batches of tissue in aluminium foil, note the mass of tissue in each batch as this will be used to calculate the volume of nuclei isolation buffer to be added in the following steps. Freeze the crosslinked tissue rapidly in liquid nitrogen.  [24]. Culture at least 1200 individual sporophytes or gametophytes at a density of six individuals per Petri dish. 2. After culture, carefully transfer the algae with dissection forceps into a sterile Erlenmeyer flask containing 400 mL of sterile seawater. Two weeks of culture should produce approximately 4-5 g (FW) of tissue.
CRITICAL STEP For some strains it may be necessary to grow more individuals per Petri dish and harvest at an earlier stage of development before cultures become fertile and produce the next generation of the life cycle. With some sporophyte strains it may be necessary to use a cell scraper to dislodge thalli from the bottom of the Petri dish. This material can be collected by filtering the seawater medium and then transferring the algal material with dissection forceps to the 400 mL of sterile seawater.

Crosslinking (Required Time: 2 h)
3. Crosslink the algal material by adding 400 mL of 2x crosslinking buffer to the 400 mL of algal material in seawater and incubating for exactly 5 min at room temperature under a chemical hood. Mix gently during the crosslinking.
CRITICAL STEP Do not incubate for more than 5 min in the 2x crosslinking buffer as this could lead to excessive crosslinking, which may interfere with the chromatin extraction and immunoprecipitation steps. Note that formaldehyde is toxic if inhaled, ingested, or absorbed through skin. 4. Filter the tissue rapidly (15 s maximum) through a sterile piece of Miracloth to eliminate the formaldehyde. Transfer the tissue to a new 50 mL tube containing 50 mL of PBS quenching buffer for 5 min at room temperature. Mix gently during quenching. 5. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes or for 10 min for sporophytes. 6. Eliminate the buffer and resuspend in 50 mL of 1x phosphate-buffered saline (PBS) to wash the tissue. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes, or for 10 min for sporophytes. 7. Remove as much of the supernatant as possible by pipetting. You can invert the tube onto a piece of Miracloth placed on absorbent paper. Wrap approximately 1 g batches of tissue in aluminium foil, note the mass of tissue in each batch as this will be used to calculate the volume of nuclei isolation buffer to be added in the following steps. Freeze the crosslinked tissue rapidly in liquid nitrogen.

PAUSE STEP
The crosslinked tissue can be stored at −80 °C at this stage, but it should be used within the next few days if possible, and should not be stored for more than 1 month.
CRITICAL STEP The filamentous structure of the material may be partly dissociated after fixation, making it difficult to recover the pellet. If this is the case, scoop the pellet out of the Falcon tube with a spatula.

Isolation of Semipure Nuclei (Required Time: 2 h)
8. Grind about 1 g of crosslinked tissue to an ultra-fine powder under liquid nitrogen using a pre-chilled mortar and pestle. Ensure that samples do not thaw during grinding.

PAUSE STEP
The crosslinked tissue can be stored at −80 • C at this stage, but it should be used within the next few days if possible, and should not be stored for more than 1 month.  [24]. Culture at least 1200 individual sporophytes or gametophytes at a density of six individuals per Petri dish. 2. After culture, carefully transfer the algae with dissection forceps into a sterile Erlenmeyer flask containing 400 mL of sterile seawater. Two weeks of culture should produce approximately 4-5 g (FW) of tissue.
CRITICAL STEP For some strains it may be necessary to grow more individuals per Petri dish and harvest at an earlier stage of development before cultures become fertile and produce the next generation of the life cycle. With some sporophyte strains it may be necessary to use a cell scraper to dislodge thalli from the bottom of the Petri dish. This material can be collected by filtering the seawater medium and then transferring the algal material with dissection forceps to the 400 mL of sterile seawater.

Crosslinking (Required Time: 2 h)
3. Crosslink the algal material by adding 400 mL of 2x crosslinking buffer to the 400 mL of algal material in seawater and incubating for exactly 5 min at room temperature under a chemical hood. Mix gently during the crosslinking.
CRITICAL STEP Do not incubate for more than 5 min in the 2x crosslinking buffer as this could lead to excessive crosslinking, which may interfere with the chromatin extraction and immunoprecipitation steps. Note that formaldehyde is toxic if inhaled, ingested, or absorbed through skin. 4. Filter the tissue rapidly (15 s maximum) through a sterile piece of Miracloth to eliminate the formaldehyde. Transfer the tissue to a new 50 mL tube containing 50 mL of PBS quenching buffer for 5 min at room temperature. Mix gently during quenching. 5. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes or for 10 min for sporophytes. 6. Eliminate the buffer and resuspend in 50 mL of 1x phosphate-buffered saline (PBS) to wash the tissue. Centrifuge at 3215 g 4 °C in an Eppendorf 5804R for 5 min for gametophytes, or for 10 min for sporophytes. 7. Remove as much of the supernatant as possible by pipetting. You can invert the tube onto a piece of Miracloth placed on absorbent paper. Wrap approximately 1 g batches of tissue in aluminium foil, note the mass of tissue in each batch as this will be used to calculate the volume of nuclei isolation buffer to be added in the following steps. Freeze the crosslinked tissue rapidly in liquid nitrogen.

PAUSE STEP
The crosslinked tissue can be stored at −80 °C at this stage, but it should be used within the next few days if possible, and should not be stored for more than 1 month.
CRITICAL STEP The filamentous structure of the material may be partly dissociated after fixation, making it difficult to recover the pellet. If this is the case, scoop the pellet out of the Falcon tube with a spatula.

Isolation of Semipure Nuclei (Required Time: 2 h)
8. Grind about 1 g of crosslinked tissue to an ultra-fine powder under liquid nitrogen using a pre-chilled mortar and pestle. Ensure that samples do not thaw during grinding.

CRITICAL STEP
The filamentous structure of the material may be partly dissociated after fixation, making it difficult to recover the pellet. If this is the case, scoop the pellet out of the Falcon tube with a spatula.

8.
Grind about 1 g of crosslinked tissue to an ultra-fine powder under liquid nitrogen using a pre-chilled mortar and pestle. Ensure that samples do not thaw during grinding.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 6 of 12 CRITICAL STEP It is important that the tissue is very thoroughly ground at this stage. To avoid cross-contamination use different mortars and pestles for different experimental conditions. 9. Transfer the powder to a 15 mL tube containing pre-chilled nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail (approximatively 5 mL of buffer for 1 g of tissue). Resuspend well by pipetting up and down. 10. Transfer the extract into a 7 mL Tenbroeck potter. Grind 10 times slowly on ice, making hemicircular movements of the potter in the tube when you insert and remove it.
To avoid cross-contamination, do not use the same Tenbroeck potter for the different CRITICAL STEP It is important that the tissue is very thoroughly ground at this stage. To avoid cross-contamination use different mortars and pestles for different experimental conditions. Methods Protoc. 2022, 5, 36 6 of 12 9.
Transfer the powder to a 15 mL tube containing pre-chilled nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail (approximatively 5 mL of buffer for 1 g of tissue). Resuspend well by pipetting up and down. 10. Transfer the extract into a 7 mL Tenbroeck potter. Grind 10 times slowly on ice, making hemicircular movements of the potter in the tube when you insert and remove it. To avoid cross-contamination, do not use the same Tenbroeck potter for the different experimental samples.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 6 of 12 CRITICAL STEP It is important that the tissue is very thoroughly ground at this stage. To avoid cross-contamination use different mortars and pestles for different experimental conditions. 9. Transfer the powder to a 15 mL tube containing pre-chilled nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail (approximatively 5 mL of buffer for 1 g of tissue). Resuspend well by pipetting up and down. 10. Transfer the extract into a 7 mL Tenbroeck potter. Grind 10 times slowly on ice, making hemicircular movements of the potter in the tube when you insert and remove it.
To avoid cross-contamination, do not use the same Tenbroeck potter for the different experimental samples.

CRITICAL STEP
To effectively break open the Ectocarpus cells, the pestle of the Tenbroeck potter should fit tightly into the cylinder, and it should be quite difficult to move the pestle down the cylinder while making the hemicircular movements. When testing a Tenbroeck potter that has not been used previously, it is important to verify cell lysis using DAPI staining (see below). Grinding is less efficient if too much tissue is extracted; do not exceed 1.5 g.
11. Incubate on ice for 20 min. Resuspend every 5 min. 12. Pre-wet two layers of Miracloth by pipetting 500 µ L of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail, and then filter the extract through the Miracloth into a 15 mL conical tube on ice. The two layers of Miracloth should be rotated at 90° to each other. Squeeze the Miracloth well to remove all liquid. This step is necessary to remove large debris. 13. Aliquot the filtered extract into several 2 mL microtubes and centrifuge for 20 min at 3000 g and 4 °C. 14. Remove the supernatant and gently resuspend and combine the pellets from the same sample in 1 mL of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail. Centrifuge the extract for 20 min at 3000 g and 4 °C. Repeat the wash with 1 mL of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail, then centrifuge the extract for 20 min at 3000 g and 4 °C. The pellet should be a pale yellow or whitish colour at this stage, and the supernatant pale brown ( Figure 2).

CRITICAL STEP
To effectively break open the Ectocarpus cells, the pestle of the Tenbroeck potter should fit tightly into the cylinder, and it should be quite difficult to move the pestle down the cylinder while making the hemicircular movements. When testing a Tenbroeck potter that has not been used previously, it is important to verify cell lysis using DAPI staining (see below). Grinding is less efficient if too much tissue is extracted; do not exceed 1.5 g.
11. Incubate on ice for 20 min. Resuspend every 5 min. 12. Pre-wet two layers of Miracloth by pipetting 500 µL of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail, and then filter the extract through the Miracloth into a 15 mL conical tube on ice. The two layers of Miracloth should be rotated at 90 • to each other. Squeeze the Miracloth well to remove all liquid. This step is necessary to remove large debris. 13. Aliquot the filtered extract into several 2 mL microtubes and centrifuge for 20 min at 3000× g and 4 • C. 14. Remove the supernatant and gently resuspend and combine the pellets from the same sample in 1 mL of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail. Centrifuge the extract for 20 min at 3000× g and 4 • C. Repeat the wash with 1 mL of nuclei isolation buffer with Triton X-100, βmercaptoethanol, and protease inhibitor cocktail, then centrifuge the extract for 20 min at 3000× g and 4 • C. The pellet should be a pale yellow or whitish colour at this stage, and the supernatant pale brown (Figure 2).
Methods Protoc. 2022, 5, x FOR PEER REVIEW 6 of 12 CRITICAL STEP It is important that the tissue is very thoroughly ground at this stage. To avoid cross-contamination use different mortars and pestles for different experimental conditions. 9. Transfer the powder to a 15 mL tube containing pre-chilled nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail (approximatively 5 mL of buffer for 1 g of tissue). Resuspend well by pipetting up and down. 10. Transfer the extract into a 7 mL Tenbroeck potter. Grind 10 times slowly on ice, making hemicircular movements of the potter in the tube when you insert and remove it.
To avoid cross-contamination, do not use the same Tenbroeck potter for the different experimental samples.

CRITICAL STEP
To effectively break open the Ectocarpus cells, the pestle of the Tenbroeck potter should fit tightly into the cylinder, and it should be quite difficult to move the pestle down the cylinder while making the hemicircular movements. When testing a Tenbroeck potter that has not been used previously, it is important to verify cell lysis using DAPI staining (see below). Grinding is less efficient if too much tissue is extracted; do not exceed 1.5 g.
11. Incubate on ice for 20 min. Resuspend every 5 min. 12. Pre-wet two layers of Miracloth by pipetting 500 µ L of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail, and then filter the extract through the Miracloth into a 15 mL conical tube on ice. The two layers of Miracloth should be rotated at 90° to each other. Squeeze the Miracloth well to remove all liquid. This step is necessary to remove large debris. 13. Aliquot the filtered extract into several 2 mL microtubes and centrifuge for 20 min at 3000 g and 4 °C. 14. Remove the supernatant and gently resuspend and combine the pellets from the same sample in 1 mL of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail. Centrifuge the extract for 20 min at 3000 g and 4 °C. Repeat the wash with 1 mL of nuclei isolation buffer with Triton X-100, β-mercaptoethanol, and protease inhibitor cocktail, then centrifuge the extract for 20 min at 3000 g and 4 °C. The pellet should be a pale yellow or whitish colour at this stage, and the supernatant pale brown (Figure 2).  15. Remove the supernatant and gently resuspend the pellets in 1 mL of nuclei isolation buffer with β-mercaptoethanol and protease inhibitor cocktail but without Triton X-100. 16. OPTIONAL STEP Verify the release of the nuclei using microscopy. Dilute 1 µL of stock solution of 2 mg/mL DAPI in water (stored at −20 • C) in 100 µL of nuclei isolation buffer without Triton X-100, and then add 2 µL of this 10x preparation to 20 µL of extract. Incubate at room temperature for 10 min, then place between a slide and coverslip and visualize under a fluorescence microscope (Figure 3).
sonication to remove debris (step 22). The top panels show the whole tube and the bottom panels the aspect of the pelleted nuclei extracts. 15. Remove the supernatant and gently resuspend the pellets in 1 mL of nuclei isolation buffer with β-mercaptoethanol and protease inhibitor cocktail but without Triton X-100. 16. OPTIONAL STEP Verify the release of the nuclei using microscopy. Dilute 1 µ L of stock solution of 2 mg/mL DAPI in water (stored at −20 °C ) in 100 µ L of nuclei isolation buffer without Triton X-100, and then add 2 µ L of this 10x preparation to 20 µ L of extract. Incubate at room temperature for 10 min, then place between a slide and coverslip and visualize under a fluorescence microscope (Figure 3). CRITICAL STEP DAPI is toxic and a mutagen. Wear gloves while working with DAPI.

Lysis of Nuclei and Sonication (Required Time: 3 h)
17. Transfer to a new 2 mL microtube and centrifuge for 20 min at 3000 g and 4 °C. Prepare the nuclei lysis buffer and ChIP dilution buffer at this stage and cool to 4 °C . 18. Remove the supernatant and resuspend the pellet in 200 µ L to 1 mL (depending on the quantity of starting tissue) of cold nuclei lysis buffer. For example, use 1 mL of nuclei lysis buffer for 1 g of tissue. 19. Keep a 5 µ L aliquot of the chromatin extract to run on an agarose gel (see below) in order to compare with the chromatin extract after sonication.
PAUSE STEP If necessary, the chromatin extract can be frozen at −80 °C overnight at this stage, but it is better to continue directly to the sonication step if possible.

Pipette 130 µ L aliquots of the chromatin extract into new, clean Covaris ® microTUBEs
(AFA Fiber Pre-Slit Snap-Cap 6 mm × 16 mm). It is important to add exactly 130 µ L to each microTUBE and to avoid foaming to ensure complete fragmentation. 21. Sonicate the chromatin extract in a Covaris ® M220 Focused-ultrasonicator™ using the following parameters: duty 25%, peak power 75, cycles/burst 200, time duration 900 s, set point temperature 6 °C (range between 4 °C and 7 °C ). Note that sonication could alternatively be carried out with another sonicator, such as the Diagenode Bioruptor Pico, with minor optimization. 22. Combine the contents of the microTUBEs after sonication and centrifuge for 5 min at 14,000 g and 4 °C to pellet the debris (Figure 2). Transfer the supernatant to a new 1.5 mL microtube. Keep 5 µ L of this sample to run on a 0.8% agarose gel (along with the unsonicated sample taken earlier) to visualize the DNA fragments after sonication. After gel electrophoresis, a smear of sonicated chromatin should be observed between 100 bp and 1000 bp (Figure 4). Note that the samples are crosslinked chromatin 15. Remove the supernatant and gently resuspend the pellets in 1 mL of nuclei isolation buffer with β-mercaptoethanol and protease inhibitor cocktail but without Triton X-100. 16. OPTIONAL STEP Verify the release of the nuclei using microscopy. Dilute 1 µ L of stock solution of 2 mg/mL DAPI in water (stored at −20 °C ) in 100 µ L of nuclei isolation buffer without Triton X-100, and then add 2 µ L of this 10x preparation to 20 µ L of extract. Incubate at room temperature for 10 min, then place between a slide and coverslip and visualize under a fluorescence microscope (Figure 3). CRITICAL STEP DAPI is toxic and a mutagen. Wear gloves while working with DAPI.

Lysis of Nuclei and Sonication (Required Time: 3 h)
17. Transfer to a new 2 mL microtube and centrifuge for 20 min at 3000 g and 4 °C. Prepare the nuclei lysis buffer and ChIP dilution buffer at this stage and cool to 4 °C . 18. Remove the supernatant and resuspend the pellet in 200 µ L to 1 mL (depending on the quantity of starting tissue) of cold nuclei lysis buffer. For example, use 1 mL of nuclei lysis buffer for 1 g of tissue. 19. Keep a 5 µ L aliquot of the chromatin extract to run on an agarose gel (see below) in order to compare with the chromatin extract after sonication.
PAUSE STEP If necessary, the chromatin extract can be frozen at −80 °C overnight at this stage, but it is better to continue directly to the sonication step if possible.

Pipette 130 µ L aliquots of the chromatin extract into new, clean Covaris ® microTUBEs
(AFA Fiber Pre-Slit Snap-Cap 6 mm × 16 mm). It is important to add exactly 130 µ L to each microTUBE and to avoid foaming to ensure complete fragmentation. 21. Sonicate the chromatin extract in a Covaris ® M220 Focused-ultrasonicator™ using the following parameters: duty 25%, peak power 75, cycles/burst 200, time duration 900 s, set point temperature 6 °C (range between 4 °C and 7 °C ). Note that sonication could alternatively be carried out with another sonicator, such as the Diagenode Bioruptor Pico, with minor optimization. 22. Combine the contents of the microTUBEs after sonication and centrifuge for 5 min at 14,000 g and 4 °C to pellet the debris (Figure 2). Transfer the supernatant to a new 1.5 mL microtube. Keep 5 µ L of this sample to run on a 0.8% agarose gel (along with the unsonicated sample taken earlier) to visualize the DNA fragments after sonication. After gel electrophoresis, a smear of sonicated chromatin should be observed between 100 bp and 1000 bp (Figure 4). Note that the samples are crosslinked chromatin CRITICAL STEP DAPI is toxic and a mutagen. Wear gloves while working with DAPI. 15. Remove the supernatant and gently resuspend the pellets in 1 mL of nuclei isolation buffer with β-mercaptoethanol and protease inhibitor cocktail but without Triton X-100. 16. OPTIONAL STEP Verify the release of the nuclei using microscopy. Dilute 1 µ L of stock solution of 2 mg/mL DAPI in water (stored at −20 °C ) in 100 µ L of nuclei isolation buffer without Triton X-100, and then add 2 µ L of this 10x preparation to 20 µ L of extract. Incubate at room temperature for 10 min, then place between a slide and coverslip and visualize under a fluorescence microscope (Figure 3). CRITICAL STEP DAPI is toxic and a mutagen. Wear gloves while working with DAPI.

Lysis of Nuclei and Sonication (Required Time: 3 h)
17. Transfer to a new 2 mL microtube and centrifuge for 20 min at 3000 g and 4 °C. Prepare the nuclei lysis buffer and ChIP dilution buffer at this stage and cool to 4 °C . 18. Remove the supernatant and resuspend the pellet in 200 µ L to 1 mL (depending on the quantity of starting tissue) of cold nuclei lysis buffer. For example, use 1 mL of nuclei lysis buffer for 1 g of tissue. 19. Keep a 5 µ L aliquot of the chromatin extract to run on an agarose gel (see below) in order to compare with the chromatin extract after sonication.
PAUSE STEP If necessary, the chromatin extract can be frozen at −80 °C overnight at this stage, but it is better to continue directly to the sonication step if possible. 20. Pipette 130 µ L aliquots of the chromatin extract into new, clean Covaris ® microTUBEs (AFA Fiber Pre-Slit Snap-Cap 6 mm × 16 mm). It is important to add exactly 130 µ L to each microTUBE and to avoid foaming to ensure complete fragmentation. 21. Sonicate the chromatin extract in a Covaris ® M220 Focused-ultrasonicator™ using the following parameters: duty 25%, peak power 75, cycles/burst 200, time duration 900 s, set point temperature 6 °C (range between 4 °C and 7 °C ). Note that sonication could alternatively be carried out with another sonicator, such as the Diagenode Bioruptor Pico, with minor optimization. 22. Combine the contents of the microTUBEs after sonication and centrifuge for 5 min at 14,000 g and 4 °C to pellet the debris (Figure 2). Transfer the supernatant to a new 1.5 mL microtube. Keep 5 µ L of this sample to run on a 0.8% agarose gel (along with the unsonicated sample taken earlier) to visualize the DNA fragments after sonication. After gel electrophoresis, a smear of sonicated chromatin should be observed between 100 bp and 1000 bp (Figure 4). Note that the samples are crosslinked chromatin PAUSE STEP If necessary, the chromatin extract can be frozen at −80 • C overnight at this stage, but it is better to continue directly to the sonication step if possible. 20. Pipette 130 µL aliquots of the chromatin extract into new, clean Covaris ® microTUBEs (AFA Fiber Pre-Slit Snap-Cap 6 mm × 16 mm). It is important to add exactly 130 µL to each microTUBE and to avoid foaming to ensure complete fragmentation. 21. Sonicate the chromatin extract in a Covaris ® M220 Focused-ultrasonicator™ using the following parameters: duty 25%, peak power 75, cycles/burst 200, time duration 900 s, set point temperature 6 • C (range between 4 • C and 7 • C). Note that sonication could alternatively be carried out with another sonicator, such as the Diagenode Bioruptor Pico, with minor optimization. 22. Combine the contents of the microTUBEs after sonication and centrifuge for 5 min at 14,000× g and 4 • C to pellet the debris (Figure 2). Transfer the supernatant to a new 1.5 mL microtube. Keep 5 µL of this sample to run on a 0.8% agarose gel (along with the unsonicated sample taken earlier) to visualize the DNA fragments after sonication. After gel electrophoresis, a smear of sonicated chromatin should be observed between 100 bp and 1000 bp (Figure 4). Note that the samples are crosslinked chromatin at this stage (i.e., not naked DNA), so precise estimation of fragment size is not possible; however, gel electrophoresis provides an approximate estimation of the efficiency of fragmentation.
at this stage (i.e., not naked DNA), so precise estimation of fragment size is not possible; however, gel electrophoresis provides an approximate estimation of the efficiency of fragmentation. CRITICAL STEP Note that the centrifugation step is essential to remove impurities that would otherwise interfere with and contaminate the immunoprecipitation step. 23. Measure the volume of supernatant and dilute tenfold with ChIP dilution buffer. For each sample, keep 50 μL of diluted, sonicated chromatin as an input control. The chromatin extract can be used directly to set up immunoprecipitations, or stored at -20 °C or −80 °C . Note that ChIP dilution buffer is also required to prepare the Dyna-Beads (Section 3.5). Store an aliquot of ChIP dilution buffer at 4 °C overnight for this.
CRITICAL STEP Note that it is important to dilute from 1% to 0.1% SDS by adding ChIP dilution buffer because a high concentration of SDS can interfere with epitopeantibody interactions.

Immunoprecipitation (Required Time: 24 h)
24. Prepare 500 μL aliquots of diluted chromatin (equivalent to 100 mg of starting tissue) in 1.5 mL Eppendorf DNA LoBind ® microtubes. Add the recommended volume of your antibody. The volume is 5 µ L for most antibodies, but optimal volumes may vary and should be determined empirically. As a negative control, add a naïve antibody, such as normal rabbit IgG CST N° 2729. As a mock control, carry out an immunoprecipitation without any antibody. Co-incubate the sonicated chromatin and antibody overnight at 4 °C on a rotating wheel (10 rpm). 25. The next day, prepare low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, TE buffer, and elution buffer. Place the first four buffers on ice to cool and pre-heat the elution buffer to 65 °C . at this stage (i.e., not naked DNA), so precise estimation of fragment size is not possible; however, gel electrophoresis provides an approximate estimation of the efficiency of fragmentation. CRITICAL STEP Note that the centrifugation step is essential to remove impurities that would otherwise interfere with and contaminate the immunoprecipitation step. 23. Measure the volume of supernatant and dilute tenfold with ChIP dilution buffer. For each sample, keep 50 μL of diluted, sonicated chromatin as an input control. The chromatin extract can be used directly to set up immunoprecipitations, or stored at -20 °C or −80 °C . Note that ChIP dilution buffer is also required to prepare the Dyna-Beads (Section 3.5). Store an aliquot of ChIP dilution buffer at 4 °C overnight for this.
CRITICAL STEP Note that it is important to dilute from 1% to 0.1% SDS by adding ChIP dilution buffer because a high concentration of SDS can interfere with epitopeantibody interactions.

Immunoprecipitation (Required Time: 24 h)
24. Prepare 500 μL aliquots of diluted chromatin (equivalent to 100 mg of starting tissue) in 1.5 mL Eppendorf DNA LoBind ® microtubes. Add the recommended volume of your antibody. The volume is 5 µ L for most antibodies, but optimal volumes may vary and should be determined empirically. As a negative control, add a naïve antibody, such as normal rabbit IgG CST N° 2729. As a mock control, carry out an immunoprecipitation without any antibody. Co-incubate the sonicated chromatin and antibody overnight at 4 °C on a rotating wheel (10 rpm). 25. The next day, prepare low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, TE buffer, and elution buffer. Place the first four buffers on ice to cool and pre-heat the elution buffer to 65 °C .
CRITICAL STEP Note that the centrifugation step is essential to remove impurities that would otherwise interfere with and contaminate the immunoprecipitation step. 23. Measure the volume of supernatant and dilute tenfold with ChIP dilution buffer. For each sample, keep 50 µL of diluted, sonicated chromatin as an input control. The chromatin extract can be used directly to set up immunoprecipitations, or stored at -20 • C or −80 • C. Note that ChIP dilution buffer is also required to prepare the DynaBeads (Section 3.5). Store an aliquot of ChIP dilution buffer at 4 • C overnight for this.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 8 of 12 at this stage (i.e., not naked DNA), so precise estimation of fragment size is not possible; however, gel electrophoresis provides an approximate estimation of the efficiency of fragmentation. CRITICAL STEP Note that the centrifugation step is essential to remove impurities that would otherwise interfere with and contaminate the immunoprecipitation step. 23. Measure the volume of supernatant and dilute tenfold with ChIP dilution buffer. For each sample, keep 50 μL of diluted, sonicated chromatin as an input control. The chromatin extract can be used directly to set up immunoprecipitations, or stored at -20 °C or −80 °C . Note that ChIP dilution buffer is also required to prepare the Dyna-Beads (Section 3.5). Store an aliquot of ChIP dilution buffer at 4 °C overnight for this.
CRITICAL STEP Note that it is important to dilute from 1% to 0.1% SDS by adding ChIP dilution buffer because a high concentration of SDS can interfere with epitopeantibody interactions.

Immunoprecipitation (Required Time: 24 h)
24. Prepare 500 μL aliquots of diluted chromatin (equivalent to 100 mg of starting tissue) in 1.5 mL Eppendorf DNA LoBind ® microtubes. Add the recommended volume of your antibody. The volume is 5 µ L for most antibodies, but optimal volumes may vary and should be determined empirically. As a negative control, add a naïve antibody, such as normal rabbit IgG CST N° 2729. As a mock control, carry out an immunoprecipitation without any antibody. Co-incubate the sonicated chromatin and antibody overnight at 4 °C on a rotating wheel (10 rpm). 25. The next day, prepare low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, TE buffer, and elution buffer. Place the first four buffers on ice to cool and pre-heat the elution buffer to 65 °C .
CRITICAL STEP Note that it is important to dilute from 1% to 0.1% SDS by adding ChIP dilution buffer because a high concentration of SDS can interfere with epitope-antibody interactions.

Immunoprecipitation (Required Time: 24 h)
24. Prepare 500 µL aliquots of diluted chromatin (equivalent to 100 mg of starting tissue) in 1.5 mL Eppendorf DNA LoBind ® microtubes. Add the recommended volume of your antibody. The volume is 5 µL for most antibodies, but optimal volumes may vary and should be determined empirically. As a negative control, add a naïve antibody, such as normal rabbit IgG CST N • 2729. As a mock control, carry out an immunoprecipitation without any antibody. Co-incubate the sonicated chromatin and antibody overnight at 4 • C on a rotating wheel (10 rpm CRITICAL STEP Take care not to over-dry the glycogen-containing pellets as they may become insoluble. CRITICAL STEP Take care not to over-dry the glycogen-containing pellets as they may become insoluble. 41. Resuspend the pellets in 30 µL of DEPC water, store the DNA at −80 • C, and use within 3 months. Traces of phenol may be detected at this stage, but they do not interfere with library construction.

DNA Analysis (Required Time: 2 h)
42. DNA concentration and size range can be analyzed using a Bioanalyzer and a High Sensitivity DNA Chip (Agilent; Figure 5). In addition, DNA concentration should be assessed using a Qubit fluorometer and a dsDNA HS Assay kit.
41. Resuspend the pellets in 30 µ L of DEPC water, store the DNA at −80 °C , and use within 3 months. Traces of phenol may be detected at this stage, but they do not interfere with library construction.

DNA Analysis (Required Time: 2 h)
42. DNA concentration and size range can be analyzed using a Bioanalyzer and a High Sensitivity DNA Chip (Agilent; Figure 5). In addition, DNA concentration should be assessed using a Qubit fluorometer and a dsDNA HS Assay kit.

Expected Results
This protocol yields approximately 20-25 μg of chromatin per gram of tissue. DNA fragment size should be between 150 bp and 1000 bp (Figure 2). To obtain strong enrichment, we recommend using between 2 μg and 5 μg of antibody and approximately 1.5 μg of chromatin per immunoprecipitation. Ideally, calibration curves should be carried out for each antibody to optimize the efficiency of immunoprecipitation and to minimize background noise. The number of immunoprecipitations required for each antibody should be determined empirically. Table 1 indicates approximate quantities of immunoprecipitated chromatin to be expected when using antibodies against different histone PTMs. At least 5 ng (we recommend 20 ng) of DNA is necessary to prepare sequencing libraries for Illumina platforms using the TruSeq ChIP Library Preparation kit (Illumina). Often, DNA immunoprecipitated using a naïve IgG is not amplifiable, as less than 1 ng can be collected by pooling six immunoprecipitations. For the test samples, we typically pool 4-5 samples per lane on an Illumina HiSeq 4000, aiming for 40-90 million reads per sample in order to obtain strong signals. Inputs should be sequenced more deeply, aiming for 100-110 million reads to accurately model the background and limit detection bias.

Expected Results
This protocol yields approximately 20-25 µg of chromatin per gram of tissue. DNA fragment size should be between 150 bp and 1000 bp (Figure 2). To obtain strong enrichment, we recommend using between 2 µg and 5 µg of antibody and approximately 1.5 µg of chromatin per immunoprecipitation. Ideally, calibration curves should be carried out for each antibody to optimize the efficiency of immunoprecipitation and to minimize background noise. The number of immunoprecipitations required for each antibody should be determined empirically. Table 1 indicates approximate quantities of immunoprecipitated chromatin to be expected when using antibodies against different histone PTMs. At least 5 ng (we recommend 20 ng) of DNA is necessary to prepare sequencing libraries for Illumina platforms using the TruSeq ChIP Library Preparation kit (Illumina). Often, DNA immunoprecipitated using a naïve IgG is not amplifiable, as less than 1 ng can be collected by pooling six immunoprecipitations. For the test samples, we typically pool 4-5 samples per lane on an Illumina HiSeq 4000, aiming for 40-90 million reads per sample in order to obtain strong signals. Inputs should be sequenced more deeply, aiming for 100-110 million reads to accurately model the background and limit detection bias.