Monitoring Chromatin Regulation in Planarians Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq)

Abstract

Planarians are an accessible model system to study animal regeneration and stem cells. Over the last two decades, new molecular techniques have provided us with powerful tools to understand whole-body regeneration and pluripotent adult stem cells specifically. We describe a method for performing Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) on planarian cells that relies on FACS to isolate different cell populations followed by immunoprecipitation and library preparation for next-generation sequencing. Whole-genome profiling of histone modifications enables a greater understanding of epigenetic mechanisms in development, pluripotency, and differentiation. This protocol adds to the growing list of functional genomic approaches to study whole-body regeneration in animals.

1 Introduction

Planarians are best known for their ability to regenerate their whole bodies and owe this remarkable ability to neoblasts, their sole population of pluripotent stem cells. Being the only dividing cell in planarians, neoblasts replace cells lost due to normal physiological turnover as well as injury [1,2,3]. Neoblasts are thus a promising model system to investigate the epigenetic regulation of pluripotency, stem cell function and differentiation, and tissue patterning during regeneration. Various studies have established conservation of key features of stem cell biology with other animals. Planarians also allow the study of stem cell heterogeneity and lineage progression from undifferentiated stem cells due to the availability of molecular markers for stem cells and their progeny [4, 5]. An advantage of using Schmidtea mediterranea as a model organism for studying epigenetics is the availability of an excellent array of genomic resources and tools to make these studies possible. These include an excellent genome assembly [6], annotations [7], genome database [8], and a transcriptome repository [9]. Nevertheless, while planarians are a promising model system for in vivo stem cell biology, we are only beginning to understand the molecular principles governing the associated regulatory mechanisms. Further research into stem cells in planarians and other model organisms will help us understand fundamental stem cell properties, including disentangling pluripotency and self-renewal [10].

Our understanding of the epigenetic control of regeneration in planarian is in its relative infancy compared to other model organisms. As the epigenetic regulation of gene expression depends on DNA methylation, histone modifications, and overall chromatin organization, understanding these in pluripotent planarian stem cells is of interest to the community [10]. With respect to DNA methylation, a number of strong lines of evidence suggest that 5-methyl cytosine (the major form of DNA methylation in animals) is not part of epigenetic regulation in planarians [10, 11]. S. mediterranea was found to have only the conserved DNA methyltransferase 2 (DNMT2) that despite its name is only thought to methylate RNA [10]. DNA methylation is read by methyl binding domain (MBD) proteins that form key components of histone modifying and chromatin remodeling complexes. In planarians, a single MBD protein, called MBD2/3, has been described. This protein actually lacks the conserved residues known to contact methylated DNA and thus is unlikely to bind 5-methyl cytosine [11]. The absence of the 5-methyl cytosine modification in the S. mediterranea genome was also confirmed in various ways, including the lack of antibody staining against 5-methyl cytosine, and undetectable levels of 5-methyl cytosine in high-performance liquid chromatography mass spectrometry [11]. These different lines of evidence suggest that the function of planarian MBD2/3 is likely independent of DNA methylation, and that DNA methylation is not involved in the epigenetic control of planarian neoblasts. The MBD2/3 protein is known to function in the Nucleosome remodeling and Deacetylase (NuRD) complex in animals. In planarians MBD2/3 and the functions of four other NuRD components have been investigated by RNAi-mediated knockdown in planarians: Smed-HDAC1 [12,13,14] Smed-CHD [15], RbAp48 [16, 17], and GATAD2 (or p66) [18]. Knockdown of each of these genes affects stem cell differentiation.

The phenotypic effects of the loss of epigenetic regulators that control gene expression can be effectively assessed during planarian regeneration using RNAi. Stem cell survival and differentiation defects can be monitored with in situ hybridization using a growing list of markers. The phenotypes observed are caused by the mis-regulation of gene expression across the genome and often, the mis-regulation of a few key genes have a large effect with respect to the observed phenotype. With the advent of Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) on planarian cells, we can now correlate the phenotypic effects with epigenetic changes at loci across the genome by measuring changes in histone marks in populations of cells as a result of RNAi. By measuring changes in histone marks that induced by RNAi experiments and correlating these changes with gene expression, we can begin to identify epigenetic targets involved in normal stem cell regulation and regeneration. So far studies have confirmed that relationships between gene expression and the enrichment of particular histone marks on nucleosomes proximal to gene promoters present in other animals are conserved [19,20,21]. For example, as in other animals, higher levels of H3K4me3 are associated with the promoters of actively transcribed genes in planarian stem cells, while H3K27me3 is associated with the promoters of silenced genes [19,20,21].

ChIP-seq was first used on whole dissociated planarians to show that the histone methyl-transferase enzymes Set1 and MLL1/2, the main mediators of H3K4me3 in animals, target markedly different genomic loci in vivo, respectively [19]. Set1 targets were shown to be associated broadly with the maintenance of basic cell function and survival, while MLL1/2 targets were specifically enriched for genes involved in ciliogenesis. These observations correlate with loss of stem cells in set1(RNAi) animals and the specific loss of cilia and associated locomotion in mll1/2(RNAi) animals. Mihaylova et al. investigated the role of planarian orthologs of a third H3K4 methyltransferase enzymes MLL3/4 [20]. In mammals, loss of MLL3/4 function has been implicated in tumorigenesis [22,23,24]. RNAi of MLL3/4 in planarians led to the formation of tumor-like outgrowths, suggesting that this histone methyl-transferase has tumor suppressor activity in planarians [20]. RNA-seq and ChIP-seq analyses performed on G2/M planarian stem cells from MLL3/4 knockdown animals indicate that genes downstream of MLL3/4 limit or promote stem cell proliferation during regeneration. The MLL3/4 protein plays a role in transcriptional regulation via mono- and/or tri-methylating H3K4 at promoters and enhancers [20] RNA-seq on the same cells revealed that a number of genes involved in cell proliferation and differentiation, including potential oncogenes, were significantly upregulated. The transcriptional changes of some genes following knockdown of planarian MLL3/4 correlate with differences in H3K4me1 peaks at the promoter region, suggestive of direct effect of MLL3/4.

A study by Dattani et al. applied an improved ChIP-seq protocol for neoblasts in S. mediterranea to generate genome-wide profiles for the active marks H3K4me3 and H3K36me3, and suppressive marks H3K4me1 and H3K27me3 [21] in order to look at epigenetic regulation of gene expression in neoblasts. As predicted from work in vertebrates and other protostomes, these marks showed conserved patterns of association with active and suppressed gene expression in planarian neoblasts. Significantly, loci that have little or no transcriptional activity in the neoblast compartment and are known to activate transcriptionally in the post-mitotic progeny during differentiation show bivalent histone modifications, with both H3K4me3 and H3K27me3 marks at promoter regions. ChIP-seq also revealed high levels of paused RNA Polymerase II at the promoter-proximal region as further evidence that these genes are bivalent in neoblasts, becoming actively transcribed upon differentiation. These findings suggest that epigenetic regulation of potency through bivalency at promoter regions is conserved across bilaterians, rather than a special feature of vertebrates [21]. Overall, these studies have established that ChIP-seq can be efficiently used in neoblasts to investigate epigenetic regulation of stem cell fate.

In this chapter, we provide step-by-step robust protocols for cell dissociation and isolation of planarian cells, chromatin extraction and sonication, immunoprecipitation , and preparation of ChIP libraries (Fig. 1). We also outline a range of quality control steps that could be used at various stages of the protocol .

Fig. 1

An overview of the ChIP-seq workflow

2 Materials

All solutions should be prepared using ultrapure water and analytical grade reagents. Reagents should be prepared and stored at the temperatures indicated. Local waste disposal regulations should be adhered to when disposing of chemical and plastic waste.

2.1 Cell Dissociation and Isolation of Stem Cells

• 1. 10× Calcium magnesium free (CMF) buffer: 25.6 mM NaH₂PO₄2H₂O, 142.8 mM NaCl, 102.1 mM KCl, and 94.2 mM NaHCO₃. Add water up to 40 mL. Mix and adjust the pH to 7.2 using NaOH (see Note 1). Make up the volume to 50 mL. Store at 4 °C.

• 2. 150 mM HEPES: 1.78 g HEPES–NaOH, pH 7.2, 40 mL H₂O (see Note 1). Make up the volume to 50 mL and store at 4 °C.

• 3. 10% (w/v) glucose: 5 g glucose, 40 mL H₂O. Make up the volume to 50 mL and store at 4 °C until used. Make fresh for each use.

• 4. 10% (w/v) BSA: 5 g BSA, 40 mL H₂O. Make up the volume to 50 mL and store at 4 °C until used. Make fresh for each use.

• 5. 0.5 M EDTA: 0.5 M EDTA, pH 8.0 (store-bought).

• 6. CMFHe²⁺: 250 μL 10% BSA, 2.5 mL 10% glucose, 5 mL 150 mM HEPES, 301 μL 0.5 M EDTA, 5 mL 10× CMF. Make up the volume to 50 mL using H₂O. Make fresh for each use (see Note 2).

• 7. Papain digestion solution: 1 mL 30 U/mL papain, 1 mL CMFHe²⁺. Make fresh for each use (see Note 3).

• 8. 5 mL round bottom tubes.

• 9. 100 μm nylon net filter.

• 10. 35 μm pore-size cell strainer cap.

• 11. 20× live DNA stain stock solution (e.g., 1 mg/mL Hoechst 34580 in distilled water). Store at −20 °C.

• 12. 2000× live cytoplasmic stain stock solution (e.g., 0.2 μg/mL Calcein AM in DMSO). Store at −20 °C.

• 13. 1000× cell viability stain stock solution (e.g., 10 μg/mL in distilled water). Store at 4 °C in a dark 1.5-mL tube to protect from light.

• 14. Planaria water: 5 g commercial sea salts (e.g., Instant Ocean) in 10 L water.

2.2 Chromatin Extraction and Sonication

• 1. 10% NP-40: Molecular biology grade 10% NP-40 solution (store-bought).

• 2. 10% Triton X-100: Molecular biology grade 10% Triton X-100 solution (store-bought).

• 3. 1 M Tris–HCl pH 7.5 solution (store-bought).

• 4. 1 M CaCl₂: 11.1 g CaCl₂ in 100 mL H₂O. Store at 4 °C.

• 5. 1 M Sucrose: 17.1 g sucrose in 50 mL H₂O. Store at 4 °C.

• 6. 0.1 M DTT: 1.5 g DTT in 10 mL of H₂O. Aliquot and store at −20 °C.

• 7. Phosphatase cocktail inhibitor 2 (Sigma-Aldrich).

• 8. Phosphatase cocktail inhibitor 3 (Sigma-Aldrich).

• 9. Protease inhibitor tablets (cOmplete protease inhibitor cocktail, Roche).

• 10. Nuclei extraction buffer (NEB): 500 μL 10% NP-40, 250 μL 10% Triton X-100, 100 μL 1 M Tris, 30 μL 1 M CaCl₂, 2.5 mL 1 M sucrose, 100 μL 0.1 M DTT, 100 μL phosphatase cocktail inhibitor 2, 100 μL phosphatase cocktail inhibitor 3, 6.32 mL H₂O. Prepare the NEB fresh every time, although stock solutions of each components can be stored. Add inhibitors just before use.

• 11. 1× Phosphate Buffer Solution (PBS): 1.86 mM NaH₂PO₄, 8.41 mM Na₂HPO₄, 175.0 mM NaCl, pH 7.4.

• 12. PBS with protease inhibitors: One protease inhibitor tablet in 50 mL cold 1× PBS. Ideally make fresh, but will keep for 2–3 days at 4 °C.

• 13. 2.5 M glycine: 9.386 g glycine in 50 mL water. 2.5 M Glycine can be made in advance and stored at room temperature (RT). Do check for microbial growth if stored for more than 7 days.

• 14. 10% SDS (store-bought).

• 15. 1 M Tris–HCl, pH 8.0 solution (store-bought).

• 16. SDS-lysis buffer: 500 μL 10% SDS, 250 μL 1 M Tris–HCl, pH 8.0, 100 μL 0.5 M EDTA, 4.15 mL H₂O. Can be made in advance and will keep for 2–3 months at RT.

• 17. ChIP buffer stock: 10 μL 10% SDS, 24 μL 0.5 M EDTA, 167 μL 1 M Tris–HCl, pH 8.0, 334 μL 5 M NaCl, 9.46 mL H₂O. Can be prepared in advance and will keep for 1–2 weeks at RT.

• 18. ChIP dilution buffer: 2 mL ChIP buffer stock, 2 μL phosphatase cocktail inhibitor 2, 2 μL phosphatase cocktail inhibitor 3, 20 μL 0.1 M DTT. Make fresh and keep on ice.

• 19. High-precision, temperature-controlled, multiple samples, in sealed tubes sonicator (e.g., Bioruptor, Diagenode).

• 20. 16% formaldehyde: 16% (w/v) formaldehyde solution (store-bought).

• 21. DNA mini-elute PCR purification kit.

• 22. DNA electrophoresis equipment (e.g., TapeStation 2200, Agilent).

• 23. DNA concentration fluorometer (e.g., Qubit, Thermo Fisher Scientific).

2.3 Immunoprecipitation and Reverse Crosslinking

• 1. Protein-A covered beads.

• 2. Magnetic separation rack for 1.5-mL tubes.

• 3. Blocking solution: 0.5% BSA in 1× PBS.

• 4. ChIP-grade antibodies: 7 μg antibody per sample (see Note 4).

• 5. Commercial Drosophila S2 chromatin as internal immunoprecipitation control (store-bought, e.g., Active Motif).

• 6. ChIP wash buffer: 50 mM HEPES–KOH, pH 8, 0.5 M LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 1 protease inhibitors tablet. Make fresh for each use.

• 7. Tris–EDTA buffer (TE): 1× TE buffer (store-bought).

• 8. 0.1× TE: 10 μL TE, 90 μL water.

• 9. TE-SDS: 2% (v/v) SDS in 1× TE.

• 10. TE/NaCl: TE buffer, 50 mM NaCl.

• 11. Elution buffer: 50 mM Tris–HCl, pH 8, 10 mM EDTA, 1% SDS.

• 12. Phenol:chloroform:isoamyl alcohol: 25:24:1 (v/v/v) phenol, chloroform, isoamyl alcohol (store-bought).

• 13. RNAse A: 100 mg/mL RNAse A solution (store-bought).

• 14. Proteinase K: 20 μg/mL proteinase K solution (store-bought).

• 15. 5 M NaCl: 29.2 g NaCl in 100 mL H₂O.

• 16. 20 mg/mL glycogen: 20 mg/mL glycogen solution (store-bought).

2.4 Preparation of ChIP Libraries for Sequencing

• 1. NEBNext Ultra II DNA Library Prep kit (NEB): End Prep Enzyme Mix, End Prep Reaction buffer, adaptor, ligation master mix, ligation enhancer, USER enzyme. PCR amplification kit: master mix, i7 primer stock solution, i5 primer stock solution.

• 2. Agencourt AMPure XP beads (Beckman Coulter).

• 3. 80% ethanol: 40 mL molecular biology grade 100% ethanol, 10 mL H₂O.

• 4. Magnetic stand-96 (e.g., Ambion, Invitrogen).

3 Methods

3.1 Dissociation and Isolation of Stem Cells

• 1. Fill a 100-mm petri dish with ice.

• 2. Compact the ice to form an even surface.

• 3. Place two filter paper circles on top of the ice.

• 4. Wrap parafilm to keep the filter paper in place.

• 5. Place an aluminum foil circle on top to complete the “stage.”

• 6. Select one 7- to 8-mm-long planarian per desired sample (see Note 5).

• 7. Place the worms on the aluminum foil.

• 8. Cut the worms using a razor blade.

• 9. Transfer the worms into a cold petri dish filled with 50 mL planaria water.

• 10. Replace planaria water with cold CMFHe²⁺ (see Note 2).

• 11. Cut the worms as small as possible with a scalpel. Wipe the scalpel frequently in order to prevent the accumulation of mucus.

• 12. Carefully transfer worm pieces to 1.5-mL tubes with a plastic Pasteur pipette. Transfer pieces with a large amount of liquid to avoid adhesion to the walls of the pipette.

• 13. Wait 10 min for the worm pieces to settle.

• 14. Remove all CMFHe²⁺.

• 15. Add 600 μL of papain digestion solution.

• 16. Incubate for 1 h at 25 °C. The solution should not be mixed, nor should the tubes be moved.

• 17. Mechanically dissociate the digested pieces by pipetting up and down using a P1000 for 20 strokes. Solution will turn cloudy.

• 18. Repeat step 17 until no large pieces are visible.

• 19. Centrifuge at 500 rcf for 5 min at 4 °C to pellet the cells.

• 20. Replace the supernatant with 1 mL of CMFHe²⁺.

• 21. Repeat steps 19 and 20.

• 22. Resuspend the pellet.

• 23. Filter the suspension through a 100-μm filter and another 35-μm filter into a 5-mL round bottom tube to remove undigested tissue fragments (see Note 6).

• 24. Add 50 μL of 20× live DNA stain stock solution and 0.5 μL of 2000× live cytoplasmic stain stock solution per 1 mL of filtered sample (see Note 7).

• 25. Incubate the samples in the dark for 1 h.

• 26. Add 1 μL of 1000× cell viability stain stock solution.

• 27. Create the proper gate for size and granularity in each cell population of interest.

• 28. Sort the cells (see Note 8) and collect cells into a tube containing ice-cold 1× PBS.

3.2 Chromatin Extraction and Sonication

• 1. Transfer cells from FACS tubes to protein low-binding 1.5-mL tubes.

• 2. Spin at 4000 rcf for 4 min at 4 °C.

• 3. Remove supernatant and pool tubes of sorted cells.

• 4. Repeat steps 2 and 3 until all of the sample is pelleted in one single tube.

• 5. Remove supernatant and completely resuspend pellet in 1 mL of NEB by gently pipetting up and down 10 times.

• 6. Add 62.5 μL 16% formaldehyde.

• 7. Leave at RT on a rotator for 7 min.

• 8. Add 50 μL of 2.5 M glycine to quench the reaction.

• 9. Leave at RT on a rotator for 3 min.

• 10. Centrifuge at 4000 rcf for 4 min at 4 °C to pellet.

• 11. Replace gently the supernatant by 1 mL of ice-cold 1× PBS with protease inhibitors, without totally resuspending it.

• 12. Centrifuge at 4000 rcf for 4 min at 4 °C to pellet.

• 13. Repeat steps 11 and 12 two more times.

• 14. Remove supernatant.

• 15. Resuspend the pellet in 120 μL cold SDS-lysis buffer by pipetting up and down.

• 16. Incubate on ice for 20 min.

• 17. Add 280 μL ChIP dilution buffer to make up the volume to 400 μL.

• 18. Sonicate the samples according to the manufacturer’s instruction to obtain average chromatin fragments between 200 and 400 bp.

• 19. Add 40 μL of 10% Triton X-100.

• 20. Centrifuge at 20,000 rcf for 15 min at 4 °C to pellet debris (see Note 9).

• 21. Carefully transfer the supernatant containing sheared chromatin to be used for immunoprecipitation to fresh 1.5 mL protein low-binding tube (see Note 10).

• 22. Aliquot 50 μL of the sample to a new low-binding tube for a test de-crosslink reaction to assess fragment distribution and concentration.

• 23. Store the sheared chromatin at 4 °C overnight or at −80 °C for longer periods.

• 24. Thaw the aliquot on ice if retrieved from −80 °C.

• 25. Add 150 μL of TE-SDS.

• 26. Heat at 65 °C for 2 h.

• 27. Purify the DNA using a mini-elute kit according to the manufacturer’s instructions (see Note 11).

• 28. Analyze the DNA fragment sizes with the DNA electrophoresis equipment (Fig. 2).

• 29. Estimate the DNA concentration using the fluorometer following manufacturer’s instructions.

Fig. 2

Example fragment size distribution analyzed on TapeStation (a) after chromatin shearing, (b) after a test de-crosslink. The optimal chromatin size distribution for ChIP-seq is between 200 and 800 bp

3.3 Chromatin Immunoprecipitation

• 1. Transfer 50 μL of beads per sample into a low-binding 1.5-mL tube.

• 2. Wash the beads thrice with 1 mL 0.5% BSA/PBS using a magnetic rack to pellet the beads.

• 3. Resuspend beads in 125 μL of blocking solution.

• 4. Add 7 μg of antibody per reaction.

• 5. Incubate tubes overnight on a rotator at 4 °C.

• 6. Wash the beads three times using 1 mL 0.5% BSA/PBS using a magnetic rack to pellet the beads.

• 7. Resuspend the beads in 50 μL of blocking solution.

• 8. Add 100 μL of sheared chromatin from the end of Subheading 3.2 to the beads per antibody sample.

• 9. Add 5 μL of Drosophila S2 chromatin spike-in. The total amount of chromatin should 1–2% of the planarian chromatin (see Note 12).

• 10. Transfer a separate 50 μL aliquot of sheared chromatin with Drosophila S2 chromatin into a low-binding 1.5-mL tube without antibodies, to be used as input DNA control.

• 11. Incubate the tubes overnight on a rotator at 4 °C.

• 12. Wash six times with 1 mL ChIP wash buffer (see Note 13).

• 13. Resuspend beads in 800 μL TE/NaCl.

• 14. Place on a rotator at 4 °C for 3 min.

• 15. Pellet the beads using a magnetic rack.

• 16. Remove all of the supernatant (see Note 14).

• 17. Resuspend the beads in 250 μL elution buffer.

• 18. Place tubes at 65 °C for 15 min on a shaking block at 1400 rpm.

• 19. Centrifuge the beads at 16,000 rcf for 1 min at RT.

• 20. Transfer the supernatant to a fresh tube.

• 21. Incubate the supernatant as well as the Input DNA control tube at 65 °C overnight for reverse crosslinking.

• 22. Add 250 μL of TE to each tube.

• 23. Add 2 μL RNAse A to each sample.

• 24. Incubate at 37 °C for 1 h.

• 25. Add 4 μL Proteinase K.

• 26. Incubate at 55 °C for 1 h.

• 27. Add 500 μL of phenol:chloroform:isoamyl alcohol.

• 28. Vortex at maximum power for 2 min.

• 29. Centrifuge at max speed at RT for 5 min.

• 30. Transfer the upper aqueous phase to a new tube avoiding contamination of residual phenol (see Note 15).

• 31. Add 2 μL of 5 M NaCl, 1.5 μL of 20 mg/mL glycogen and 1.5 mL of −20 °C 100% ethanol.

• 32. Incubate at −80 °C for 1 h.

• 33. Centrifuge at max speed for 30 min at 4 °C.

• 34. Replace the supernatant with 1 mL 70% ethanol.

• 35. Centrifuge at max speed for 10 min at 4 °C.

• 36. Remove most of the supernatant with disturbing the bottom of the tube.

• 37. Leave the open tubes at room temperature for 1 h to dry.

• 38. Resuspend the DNA in 50 μL 1× TE.

• 39. Shake the tubes at 37 °C for 20 min.

• 40. Incubate the tubes at RT for 45 min to completely resuspend the DNA.

3.4 Preparation of ChIP Libraries for Sequencing

• 1. Transfer 50 μL of fragmented DNA to a PCR tube.

• 2. Add 3 μL NEBNext Ultra II End Prep Enzyme Mix and 7 μL NEBNext Ultra II End Prep Reaction buffer.

• 3. Pipette up and down at least 10 times 50 μL using a P200 to thoroughly mix the solution.

• 4. Briefly spin the tubes for 5 s at max speed to collect liquid from the sides of the tubes.

• 5. Place in a thermocycler with a heated lid.

• 6. Run a program at 20 °C for 30 min followed by 65 °C for 30 min.

• 7. Dilute the NEBNext Adaptor in Tris/NaCl, pH 8.0 according to the DNA concentration previously measured (see Note 16).

• 8. Add 2.5 μL of diluted adaptors to the reaction mix.

• 9. Add 30 μL of ligation master mix and 1 μL of ligation enhancer to the reaction mix.

• 10. Pipette up and down at least 10 times 80 μL using a P200 to thoroughly mix the solution.

• 11. Briefly spin the tubes 5 s at max speed to collect all the liquid from the sides.

• 12. Incubate at 20 °C for 15 min in a thermocycler with the heated lid off.

• 13. Add 3 μL of USER enzyme to the mixture.

• 14. Mix well and incubate at 37 °C for 15 min in a thermocycler with the heated lid set to 47 °C.

• 15. Samples can be stored at −20 °C overnight at this point.

• 16. Allow AMPure XP beads and the DNA samples to warm to RT for approximately 30 min.

• 17. Vortex the beads for 5 s at max speed.

• 18. Add 87 μL of the beads to the ligation mix.

• 19. Mix well by pipetting up and down at least 10 times.

• 20. Incubate the mix for 5 min at RT.

• 21. Transfer to a 96-well PCR plate.

• 22. Place the plate on a magnetic rack.

• 23. Wait for 5 min for the beads to separate from the supernatant.

• 24. Remove the supernatant carefully without disturbing the beads.

• 25. Add 180 μL of freshly prepared 80% ethanol to the tube while it is on the rack. 80% ethanol is added to the beads slowly, and the beads are not to be resuspended in it.

• 26. Incubate on the rack at RT for 30 s.

• 27. Carefully remove and discard the supernatant without disturbing the beads.

• 28. Repeat steps 25–27 once. Be sure to remove all of the supernatant (see Note 17).

• 29. Air dry the beads for 5 min on the magnetic rack.

• 30. Remove the plate from the magnetic rack.

• 31. Add 17 μL 0.1× TE to each well.

• 32. Resuspend the beads fully by pipetting up and down 10 times.

• 33. Incubate the beads off the magnetic rack for 3 min at RT.

• 34. Place the plate on the magnetic rack for 5 min.

• 35. Transfer 15 μL of the supernatant to a fresh PCR plate.

• 36. Samples can be stored at −20 °C at this point.

3.5 PCR Enrichment and Purification of DNA

• 1. Allow samples to warm to RT for approximately 30 min.

• 2. Add 25 μL of Master mix, 5 μL of i7 primer stock solution, and 5 μL of i5 primer stock solution to each sample.

• 3. Pipette up and down at least 10 times 80 μL using a P200 to mix the solution thoroughly.

• 4. Place the tube on a thermocycler.

• 5. Perform PCR amplification as follows:

  Initial denaturation at 98 °C for 30 s/N [denaturation at 98 °C for 10 s, annealing/extension at 65 °C for 75 s] followed by final extension at 65 °C for 5 min. N = 3–15 (see Note 18).

• 6. Allow AMPure XP beads to warm at room temperature for approximately 30 min.

• 7. Vortex the beads for 5 s at max speed.

• 8. Add 45 μL of beads to the ligation mix.

• 9. Pipette up and down at least 10 times.

• 10. Incubate the beads for 5 min at room temperature.

• 11. Place the plate on a magnetic rack and allow the beads to separate from the supernatant.

• 12. Wait for 5 min.

• 13. Repeat step until the solution is clear.

• 14. Carefully remove and discard the supernatant.

• 15. Add 180 μL of freshly prepared 80% ethanol to the tube while it is on the stand. 80% ethanol is added to the beads slowly, and the beads are not to be resuspended in it.

• 16. Incubate on the magnetic rack at room temperature for 30 s.

• 17. Carefully remove and discard the supernatant without disturbing the beads.

• 18. Repeat steps 6–8 once. Be sure to remove all of the supernatant (see Note 17).

• 19. Air dry the beads for 5 min on the magnetic rack.

• 20. Remove the plate from the magnetic rack.

• 21. Add 35 μL of 0.1× TE to the beads to elute the DNA target.

• 22. Resuspend the beads fully by pipetting up and down 10 times.

• 23. Incubate the beads off the magnetic rack for at least 2 min at room temperature.

• 24. Place the plate back to the magnetic rack.

• 25. Wait for 5 min.

• 26. Repeat step until the solution is clear.

• 27. Transfer 30 μL of the supernatant to a fresh tube.

• 28. Aliquot the samples and/or dilute for different analysis (see Note 19).

• 29. Check the size distribution using the DNA electrophoresis equipment (Fig. 3).

• 30. The samples may need to be diluted before loading after normalizing concentrations of different libraries using a qPCR-based library quantification kit.

• 31. Process samples for paired-end sequenced on an Illumina NextSeq or other Illumina machine.

Fig. 3

Size distribution of (a) input along with 3 ChIP DNA libraries, (b) ChIP DNA library

4 Notes

• 1. Set pH of 10× CMF and HEPES very carefully, an extra drop of NaOH easily shoots the pH up.

• 2. Instead of CMFHE²⁺, Holtfreter’s solution diluted 5/8 in distilled water (5/8 Holtfreter; 21.88 g NaCl, 0.63 g CaCl₂, 0.31 g KCl, 1.25 g NaHCO₃ in 10 L distilled water, pH 7.4) or PBS containing 1 mM EDTA can also be used.

• 3. Papain can be substituted by other proteinases, such as 0.25% (w/v) trypsin or 1 mg/mL collagenase in CMFHE²⁺. Digestion times will vary with different enzymes and their concentrations, and therefore digestion time must be standardized.

• 4. Typically, 3–7 μg of antibody is required for every 25 μg of chromatin. Using an optimal concentration of antibody can significantly reduce background. The amount of antibody required could be titrated by performing a ChIP experiment using different antibody concentrations. The different antibodies tested in our laboratory include H3K9ac, H3K27ac, H3K9me3, H3K4me3, H3K36me3, H3K4me1, H3K27me3, and RNAPII-Ser5P.

• 5. Select 30–40 of wild-type (a week starved) [25] or experimental (RNAi, irradiated, etc.) worms. The number of worms used depends on number of cells required for each experiment. Typically, at least 0.6–0.7 million stem cells are obtained per FACS session using 40 animals. Chromatin from these cells is used for 4 ChIP reactions (3 ChIP for histone marks antibodies with an input control).

• 6. Filtering the cell suspension removes any large debris and enriches neoblasts and neoblast progeny.

• 7. The amount of stain necessary will vary based on the number of cells/worms used. This must be worked out and the same amount should be added for all of the samples that are used in each experiment.

• 8. The first time the cell dissociation protocol is performed, it is advisable to check the cell suspension under a fluorescent microscope to confirm cell viability and optimal dissociation.

• 9. As the pellet can be small, the supernatant must be removed carefully without aspirating the pellet.

• 10. The cell debris in the pellet can be re-suspended in 400 μL ChIP dilution buffer and used in parallel as control in the test de-crosslink reaction.

• 11. It is important to determine the chromatin concentration for normalizing samples at the immunoprecipitation step, and hence, a test de-crosslink is performed.

• 12. Commercial Drosophila S2 chromatin is added as spike-in. Alternatively, S2 cells can be added before chromatin preparation. Drosophila S2 spike-in is added to the chromatin before immunoprecipitations simply as a method to normalize any technical differences in immunoprecipitations across replicate libraries [26].

• 13. Beads are often stuck on the tube caps, and the samples can be centrifuged very briefly to collect all the beads at the bottom of the tube.

• 14. If all the supernatant could not be removed, tubes can be centrifuged at 2000 rcf for 3 min at 4 °C to remove all the TE.

• 15. To avoid contamination of phenol, transfer the aqueous layer multiple times in small volumes. One can start with a P100 and then move to P20 or P10 to transfer the liquid.

• 16. This is based on the DNA concentration values from the end of Subheading 3.2. For 1 μg to 101 ng input, no adaptor dilution (15 μM final concentration), for 100 ng to 5 ng, 1:10 adaptor dilution (1.5 μM final concentration), for <5 ng, 1:25 adaptor dilution (0.6 μM final concentration).

• 17. Briefly spin the plate if necessary, place back on the magnetic stand, and remove traces of ethanol with a P10.

• 18. The number of cycles depends on DNA input and sample type. The number of cycles should be high enough to provide sufficient library fragments required for a successful sequencing run, but low enough to avoid PCR artefacts and over-cycling. The number of PCR cycles recommended can be found in Table 1 and serve as a starting point to determine the number of PCR cycles best for standard library preparation.

• 19. Qubit, agarose gel, TapeStation or bioanalyzer can be used to test the suitability of the libraries for sequencing.

Table 1 Scaling of PCR amplification cycles based on input DNA. The ideal number of PCR cycles to amplify libraries for sequencing depends on the amount of DNA that goes into the end repair reaction (Subheading 3.4). In our experience, this can vary depending on the antibody used and the amount of chromatin used in immunoprecipitation