title: GSM9160475: ES_LmnaKO_shCbs_H3K9ac_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: ESCs were dissociated by 0.05% trypsin-EDTA followed by pre-plating to remove feeders. 1 million ESCs were cross-linked with 1% formaldehyde (Sigma-Aldrich, F8775) at room temperature for 10 minutes and fixation was quenched by addition of glycine to a final concentration of 125 mM for 10 minutes on ice. Cells were collected and washed three times with ice-cold PBS, and then resuspended in cell lysis buffer (50mmol/L Tris pH 8.0, 2 mM EDTA pH 8.0, 0.1% NP-40, 10% glycerol) supplemented with protease and phosphatase inhibitors (Merck, 535142 and 524632). After 10 minutes of incubation on ice, the nuclei were pelleted at 960xg for 5 minutes and lysed in nuclei lysis buffer (50mmol/L Tris pH 8.0, 1% SDS, 5mmol/L EDTA pH 8.0) supplemented with protease and phosphatase inhibitors for 10 minutes at room temperature. Lysates were then sonicated with Bioruptor® Plus (Diagenode) with high power mode for 45 cycles (sonication cycle: 30 sec ON, 30 sec OFF) to shear chromatin to 200–500 bp in nuclear lysis buffer. After centrifugation, the supernatant was diluted 1:10 with ChIP dilution buffer (50mmol/L Tris pH8.0, 200mmol/L NaCl, 5mmol/L EDTA pH 8.0, 0.5% NP40) with protease and phosphatase inhibitors and pre-cleaned with Protein G Sepharose beads (GE Healthcare, 17061801) for 1 hour at 4°C while rotating.  The diluted lysates were incubated with 1µg H3K9ac (Cell Signaling Technology, 9649S), H3K27ac (Active motif, 39135), H3K9me3 (Abcam, ab8898) antibodies overnight followed by incubation with BSA-coated protein G sepharose. The immunoprecipitate was washed three times for 5 minutes each with NaCl wash buffer (20mmol/L Tris pH 8.0, 500mmol/L NaCl, 2mmol/L EDTA pH 8.0, 0.1% SDS, 1% NP-40), LiCl wash buffer (20mmol/L Tris pH 8.0, 500mmol/L LiCl, 2mmol/L EDTA pH 8.0, 0.1% SDS, 1% NP40) and TE wash buffer (20mmol/L Tris pH 8.0, 2mmol/L EDTA pH 8.0). Elution buffer (20mmol/L Tris pH 8.0, 2mmol/L EDTA pH 8.0, and 2% SDS) was used to extract protein/DNA complexes from protein G Sepharose beads. After reverse crosslinking at 65 °C overnight, proteinase K digestion at 55 °C for 1h was performed and DNA was purified with the ChiP DNA Clean&Concentrator kit (D5205, Zymo Research). The size of sheared chromatin was assessed by Bioanalyzer 2100 (Agilent). NGS libraries were prepared with NEBNext® Ultra™ II DNA Library Prep Kit from Illumina (E7645s, New England BioLabs).