GSM3379697: HepG2 ChIPSeq Ctrl LMNB; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
LMNB1
Cell type
Cell type Class
Liver
Cell type
Hep G2
Primary Tissue
Liver
Tissue Diagnosis
Carcinoma Hepatocellular
Attributes by original data submitter
Sample
source_name
hepatocarcinoma (HepG2) cell
tissue
HepG2 cells
cell type
hepatocarcinoma
treatment
None
chip-antibody
Abcam rabbit polyclonal Ab16048
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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.