GSM5394155: WT2_me3; Mus musculus; ChIP-Seq
Immortalized MEFs isolated from HAT1+/+ (n= 2) and HAT1-/- (n=2) embryos were collected, washed with PBS, and resuspended in CiA fixing buffer (50 mM HEPES, pH 8, 1 mM EDTA, 100 mM NaCI, and 0.5 mM EGTA). Cells were fixed with 1% formaldehyde (Thermo Scientific) for 10 min at room temperature. Crosslinking reaction was quenched with 0.125M glycine. Cells were washed with PBS supplemented with 1X of Complete protease inhibitor cocktail (Roche), pelleted and frozen. The pellets were thawed and resuspended 0.8mL of ice-cold TE buffer (pH 8.0) supplemented with 1X of Complete protease inhibitor cocktail (Roche). Human Dbt immortalized myoblasts were 30 added to the mouse cells to be used as a spike-in control. Samples were sonicated with an Active Motif sonicator for 20 min at 30% amplitude using a 30sec on/off cycle. Sheared chromatin samples were adjusted to RIPA 200 mM NaCl buffer (final concentrations of 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 200 mM NaCl in 1X TE). Samples were incubated on ice for 5 minutes and centrifuged at 13,000 rpm for 10 min at 4°C. H3K9me2 and H3K9me3 antibodies (Abcam ab1220 and ab8898, respectively) were added to the supernatant, and the samples were incubated at 4°C with rotation for 2 hours. Next, pre-washed with RIPA 200mM NaCl buffer Dynabeads (Invitrogen) were added to the samples for overnight incubation. After incubation, Dynabeads were isolated and washed twice with RIPA buffer (0.1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100 in 1X TE), twice with RIPA 200 mM NaCl Buffer, twice with LiCl Buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate in 1X TE), and twice with TE buffer. Beads were isolated, sonicated DNA was decrosslinked overnight and eluted using the MinElute PCR kit (Qiagen). Sequencing libraries were prepared by PCR amplification and sequenced with pairedend reads by Novogene.