GSM4879071: Crypt-H3K9me2; Mus musculus; ChIP-Seq
Total cell extracts were prepared in S300 buffer (20 mM Tris pH 8.0, 300 mM NaCl, 10%Glycerol, 0.2% Igepal). After 20 min incubation on ice, following centrifugation (13000 rpm,10 min, 4°C), pellets containing endogenous histones were lyzed in Urea Buffer (25 mM TrispH 6.8, 6 M Urea, 1 mM EDTA, 10% glycerol), sonicated and boiled immediately with SDSLaemmlisample buffer. Cytosolic and nuclear extracts were prepared with Nuclear PrepBuffer (10 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgCl, 300 mM Sucrose, 0.2% Igepal) onice for 5 min. Nuclei were isolated by centrifugation (2500 rpm, 10 min, 4°C). Nuclei werewashed once in Nuclear Prep Buffer, lysed in S300 buffer, incubated on ice for 20 min andcentrifuged for 10 min at 13000 rpm at 4°C. Protease inhibitors were omitted when used forin vitro assays. 250 mg of chromatin supplemented with 5% spike-in of S2 Drosophila chromatin (prepared in thesame manner) and 5 mg of antibodies were used. The next day, chromatin lysates were incubated for 4 hours with protein-G Sepharose beads (GE Healthcare). Beads were washed 3 3 with low-salt buffer (150 mM NaCl, 20 mM Tris-HCl pH 8, 2 mM EDTA, 0.1%SDS, 1% Triton X-100) and 1 3 with high-salt buffer (500 mM NaCl, 20 mM Tris-HCl pH 8, 2 mM EDTA, 0.1% SDS, 1% Triton X-100),and then re-suspended in de-crosslinking solution (0.1 M NaHCO3, 1% SDS). DNA was purified with QIAquick PCR purification kite4 Molecular Cell 77, 840–856.e1–e5, February 20, 2020(QIAGEN) according to manufacturer's instructions. DNA libraries were prepared with 2–10 ng of DNA using an in-house protocol(Blecher-Gonen et al., 2013) by the IEO genomic facility and sequenced on an Illumina HiSeq 2000.