title: GSM5653835: H3K27me1_ChIP-seq_mESC_DNMT_TKO; Drosophila melanogaster; Mus musculus; ChIP-Seq
library_construction_protocol: About 10 million cells per cell line were grown and directly crosslinked on the plate with 1% formaldehyde (Sigma) for 10 minutes at room temperature and the reaction was stopped using 125nM Glycine for 5 minutes. Fixed cell preparations were washed with ice-cold PBS, scraped off the plate, pelleted, washed twice again in ice-cold PBS, and flash frozen pellets stored at −80°C. Thawed pellets were resuspended in 500ul cell lysis buffer (5 mM PIPES-pH 8.5, 85 mM KCl, 1% (v/v) IGEPAL CA-630, 50 mM NaF, 1 mM PMSF, 1 mM Phenylarsine Oxide, 5 mM Sodium Orthovanadate, EDTA-free Protease Inhibitor tablet) and incubated 30 minutes on ice. Samples were centrifugated and pellets resuspended in 500ul of 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 and EDTA-free protease inhibitor tablet) and incubated 30 minutes on ice. Sonication of lysed nuclei was performed on a BioRuptor UCD-300 at max intensity for 60 cycles, 10 s on 20 s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis of a reversed cross-linked and purified aliquot. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and concentrated using Nanosep 10k OMEGA (Pall). Before ChIP reaction 2% of sonicated Drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing. ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit for Histones. Dynabeads Protein A (Invitrogen) were washed, then incubated with specific antibodies, 1.5 million cells of sonicated cell lysate, and protease inhibitors for 10 hr, followed by 20 min wash cycle using the provided wash buffers (Diagenode Immunoprecipitation Buffers, iDeal ChIP-seq kit for Histone). Reverse cross-linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail at 65°C for 30 min followed by 2ul Proteinase K at 65°C for 30 min. Samples were then purified with QIAGEN MinElute PCR purification kit (QIAGEN) as per manufacturers' protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse cross-linked and DNA was isolated following the same protocol. KAPA Hyper Prep Kit