GSM2609318: c-iPSC FPLD D4; Homo sapiens; RNA-Seq
For RNA-seq, total RNA was isolated using the Ambion TRIzol® Reagent RNA extraction kit (Life Technologies), quality checked using a BioAnalyzer 2100 and processed for library preparation. For ChIP, fibroblasts (10e7 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 10e7 cell-equivalents) was diluted 10 times in RIPA buffer without SDS, and incubated on a rotator overnight at 4oC with the primary antibody. For "LMNA ChIPs", 20 µg anti-lamin A/C antibody (Santa Cruz, cat# sc-7292 ) was used and pre-coupled to magnetic Dynabeads Protein G (Invitrogen). For "LMNB1 ChIPs", an anti-lamin B1 antibody was used (Abcam, cat# 16048; rabbit polyclonal) and pre-coupled to magnetic Dynabeads Protein G (Invitrogen). The 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. RNA-seq library was prepared according to the Illumina protocol, and sequenced on an Illumina NextSeq500 at the Norwegian Sequencing Center. ChIP-seq libraries were prepared according to the Illumina protocol, and sequenced on an Illumina HiSeq4000 at the Norwegian Sequencing Center.