GSM5221279: ESC input repl2; Mus musculus; ChIP-Seq
1% formaldehyde was added to cultured cells to cross-link proteins to DNA. Isolated nuclei were then lysed with lysis buffer (50mM Tris-HCl, pH 8.1, 10 mM EDTA, pH 8, 1% SDS, 1X protease inhibitor cOmplete EDTA-free cocktail, Roche). Nuclei were sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and incubated overnight with the ChIP-grade antibodies against H3K9me2 and H3K9me3. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon proteinase K digestion (50°C for 3 h) and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform, ethanol precipitated and quantified by qPCR. The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) was used in the following steps. ChIP samples (1 ng) was end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).