title: GSM4830170: input_ESC_DMSO_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were detached with TrypLE, washed once with PBS, fixed with 1 % formaldehyde for 10 minutes in room temperature followed by quenching with 125 mM glycine. For each sample, three million mESCs and 750 000 S2 cells were lysed in LB1 (50 mM HEPES-KOH, 140 nM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % Igepal ca-630 and 0.25 % Triton X-100, 5 mM Na-butyrate and 1x protease inhibitor cocktail) and collected by sentrifugation. Lysis was continued in LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 5 mM Na-butarate and 1 x protease inhibitor cocktail) followed by sentrifugation, nuclei were lysed in LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 % Na-deoxycholate, 0.5 % N-Lauroylsarcosine, 5 mM Na-butyrate and 1x protease inhibitor cocktail) and chromatin fragmented with Bioruptor NextGen for 35 cycles (high-setting). Lysates were clarified and protein-DNA complexes were isolated with ab4729 (Abcam) antibody for H3K27Ac and ab8898 (Abcam) antibody for H3K9me3 and Protein A Dynabeads (Invitrogen). 10 % of sample was taken as input before adding the antibody and stored in +4 C until de-crosslinking step. Beads were washed 7 times with RIPA buffer (50 mM HEPES-KOH pH7.5, 1 mM EDTA, 1 % Igepal CA-630, 0.7 % Na-deoxycholate, 500 mM LiCl, 5 mM Na-butyrate and 1 x protease inhibitor cocktail), once with TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA and 50 mM NaCl) and eluted from beads with Elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1% SDS). Samples were decrosslinked for 16 h in +65 C with 550 mM NaCl and proteinase K, treated with RNAseA and DNA was extracted with Phenol:Chloroform:Isoamylalcohol followed by chloroform extraction and precipitation with 30 µg glycogen, 0.3 M Na-acetate and ethanol. 10 ng of DNA was used to prepare sequencing libraries with KAPA HyperPrep Kit (Roche) according to manufacturer's instructions.