title: GSM6170907: H3K9me3_Atf7ipKO; Mus musculus; ChIP-Seq
library_construction_protocol: Approximately 15 x 10^6 cells were harvested per IP and fixed with 1 % methanol-free formaldehyde for 8 min. Cross-linking was quenched by adding glycine to a final concentration of 0.125 nM and incubated for 10 min at 4 ˚C on ice. Cells were pelleted at 600 x g for 5 min, washed with cold PBS and incubated for 10 min on ice in a buffer containing 10 mM EDTA, 10 mM Tris pH 8, 0.5 mM EGTA and 0.25 % Triton X-100. After centrifugation, cells were incubated in 1 mM EDTA, 10 mM Tris pH 8, 0.5 mM EGTA and 200 mM NaCl for 10 min on ice. Chromatin was extracted in a high salt buffer (HSB) containing 50 mM HEPES pH 7.5, 1 mM EDTA, 1 % Triton X-100, 0.1 % deoxycholate, 0.2 % SDS and 500 mM NaCl for 2 hours at 4 ˚C and chromatin was sheared using a Bioruptor Pico sonicator (Diagenode). 100 µg of chromatin was used per IP reaction with 30 µl of pre-blocked magnetic Protein A beads (Invitrogen). Beads were blocked with 1 mg of BSA and 100 ng of yeast tRNA (Sigma) in TE buffer containing proteinase inhibitor mix (Roche) before use. Prior to the IP, chromatin was pre-cleared with 20 µl of blocked beads for 1 hour at 4 ˚C. 5 % of input material was kept at -20 ˚C and de-crosslinked along the IP material. 5 µg of antibody was used per IP for overnight incubation at 4 ˚C. The next day, 30 µl of blocked beads were added to the chromatin and incubated for 4h at 4 ˚C. Beads were separated on a magnet and washed twice for 8 min with HSB, one time with 50 mM LiCl, 0.5 % NP-40, 0.5 % deoxycholate, 1 mM EDTA, and 10 mM Tris pH 8. After two additional washes with TE for 8 min, chromatin was eluted after 30 min incubation at 37 ˚C with 60 µg RNaseA (Roche) in 1 % SDS and 100 mM NaHCO3, followed by 3 h incubation adding 10 mM EDTA, 40 mM Tris pH 8 and 60 µg of Proteinase K (Roche). Final de-crosslinking was done overnight at 65 ˚C. Eluted material was cleaned up using phenol chloroform extraction and quantified using a Qubit 2.0 fluorometer (Thermo Scientific). The following antibodies were used for ChIP: H3K9me3 (Abcam ab8898 ). ChIP-seq library using NEBNext ChIP-seq Library Prep Master Mix set for Illumina (NEB E6240)