title: GSM3379699: HepG2_ChIPSeq_CsA_LMNB; Homo sapiens; ChIP-Seq
library_construction_protocol: ChipSeq: ChIP was performed as previously described (Rønningen et al., 2015). Untreated and CsA treated HepG2 cells were cross-linked with 1% formaldehyde for 10 min, with 5 x106 cells per condition. The cells were lysed for 10 min in RIPA lysis buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% triton X-100, 1% SDS, 0.1% sodium deoxycholate, 1 mM PMSF, and protease inhibitors). To generate 200-500bp DNA fragments the cells were sonicated 4 times for 10 min in a Bioruptor (Diagenode). The samples were sedimented at 10000g for 10 min and the supernatant was diluted 10 times 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% sodium deoxycholate, 1 mM PMSF, and protease inhibitors). Part of the diluted chromatin were kept for input; the remaining chromatin was incubated overnight at 4°C with primary antibodies coupled to magnetic beads DynaBeads Protein G (Invitrogen). The antibodies used were mouse anti-Lamin A/C (10µg, sc7292x, Santa Cruz Biotechnology) and rabbit anti-LaminB1 (10µg, ab16048, Abcam). The ChIP samples were washed three times in ice-cold-RIPA buffer. The samples were incubated for 6 hours at 68°C in elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS) and 40 ng of proteinase K, for crosslink reversal an DNA elution. The DNA was extracted and dissolved in H2O and processed for Illumina library preparation and sedquencing. The ChIP-seq and RNA-seq sequencing libraries were prepared according to  Illumina protocols for the HSeq2500 at the Norwegian Sequencing Center.
attributes.tag: GEO Accession
attributes.value: GSM3379699