GSM1313399: ADF04DF_LMNA_rep1; Homo sapiens; ChIP-Seq
Cells (107 per ChIP) were cross-linked in suspension for 10 min in PBS containing 1% formaldehyde before quenching with 1.25 mM glycine. Cells were lysed for 30 min at 4oC on a rotator in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 1 mM PMSF, 1x protease inhibitor cocktail) adjusted to 1% SDS, and sonicated for 3 times 15 min in a Bioruptor (Diagenode) with 30 sec ON/OFF at high power to generate chromatin fragments of ~200-400 base pairs (bp). After sedimentation, chromatin (supernatant from 107 cell-equivalents) was diluted 10 times in RIPA buffer without SDS, and incubated on a rotator overnight at 4oC with 50 µg anti-lamin A/C antibody (Santa Cruz sc-7292) pre-coupled to magnetic Dynabeads Protein G (Invitrogen). An irrelevant mouse IgG was used as control. ChIP material was collected and washed 3 times in 1 ml of ice-cold RIPA buffer without protease inhibitors. The crosslink was reversed and DNA eluted for 6 h on a shaker at 37oC in elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS) containing 0.5 µg/ml RNase A and 2 µg/ml Proteinase K). DNA was purified as described, processed for library preparation. The sequencing library was prepared according to the Illumina protocol for the HSeq2500 at the Norwegian Sequencing Center.
GEO Accession
GSM1313399