title: GSM2166098: HSF_FPLD-p1_expression; Homo sapiens; RNA-Seq
library_construction_protocol: 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.