GSM5990198: M-1751_CC408_G34R_P7_crx_H3K27ac; Mus musculus; ChIP-Seq
Crosslinked ChIP-seq   Crosslinked ChIP-seq was performed as described previously (Harutyunyan et al., 2019). Briefly, 30-50mg pulverized tissue fixed with 1% formaldehyde (Sigma), followed by reverse crosslinking with glycine. Fixed tissues were then pelleted and stored at −80 °C until use. To isolate nuclei from tissue, pellets were dounced briefly with syringe and needle (25 5/8 gauge) prior to incubation with nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS, 50 mM NaF, 1 mM PMSF, 1 mM Phenylarsine Oxide, 5 mM Sodium Orthovanadate) for 30 mins on ice. Sonication of lysed nuclei was performed on a BioRuptor (Diagenode) for 20 cycles of 30s and 300s off. Samples were checked for sonication efficiency using the criteria of 150–500 bp by gel electrophoresis.   ChIP reaction for DNMT3A was performed as follows: anti-DNMT3A (1:250, ab2850), were incubated with 35 ul protein A Dynabeads at 4 °C for 3 h, then chromatin from ~1-4 million cells was added in RIPA buffer, incubated at 4 °C o/n, washed using buffers from Ideal ChIP-seq Kit (one wash with each buffer, corresponding to RIPA, RIPA + 500 mM NaCl, LiCl, TE), eluted from beads by incubating with Elution buffer for 30 min. at room temperature.   Reverse cross linking took place on a heat block at 65°C for 4 h. ChIP samples were then treated with 2µl RNase Cocktail at 65°C for 30 min followed by 2µl Proteinase K at 65°C for 30 min. Samples were purified with QIAGEN MiniElute PCR purification kit. In parallel, input samples (chromatin from 50,000 cells) were reverse crosslinked, and DNA isolated following the same protocol.    Native ChIP-seq   For histone post-translational modifications, native ChIP were performed as described previously (Chen et al., 2018) with minor adjustments. Briefly, 30-50mg pulverized tissue was dissociated passing through a syringe (25 5/8 gauge) 20x in douncing buffer (10mM Tris Cl pH7.5, 4mM MgCl2, 1mM CaCl2, and protease inhibitor cocktail (Roche)). Disassociated cells were then digested with 30U of micrococcal nuclease (Worthington) at 37°C for 7 mins, yielding predominantly mono- and di-nucleosomes. Cells were then lysed in ice-cold hypotonic lysis buffer (0.2 mM EDTA, 0.1 mM benzamidine, 0.1 mM PMSF, 1.5 mM DTT, and protease inhibitor cocktail (Roche)) to yield chromatin. Chromatin was then incubated with pre-conjugated antibody:bead complex containing H3K27me3 (CST 9733, 4 μL / IP), H3K27ac (CST 8173, 4 μL / IP), H3K36me3 (Active Motif 61022, 4 μL / IP), or H3K36me2 (Active Motif 39255, 4 μL / IP) complexed to Protein A Dynabeads (Invitrogen, 20 μL / IP) or Anti-Mouse Dynabeads (Invitrogen, 40 μL / IP) rotating at 4°C overnight. Beads were then washed twice in ChIP Wash Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and protease inhibitor cocktail) and once more in Final Wash Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and protease inhibitor cocktail). DNA was then eluted in 100mM NaHCO3 and 1% SDS at 65°C for 2h, and purified using phenol:choloroform extraction followed by ethanol precipitation. ChIP library preparation was carried out using KAPA HTP Illumina library preparation reagents (Roche), following manufacturer's instructions. Briefly, 50 µl of ChIP DNA sample was incubated with 45 µl end repair mix at 20 °C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50 µl buffer enzyme mix at 30 °C for 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead-bound sample was incubated with 45 µl buffer enzyme mix and 5 µl of TruSeq DNA adapters (Illumina), for 20 °C 15 min, followed by Ampure XP bead purification. Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using gel excision to collect 200–700 bp fragments.