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| Status |
Public on Feb 19, 2019 |
| Title |
HepG2_ChipSeq_Ctrl_LMNA |
| Sample type |
SRA |
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| Source name |
hepatocarcinoma (HepG2) cell
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| Organism |
Homo sapiens |
| Characteristics |
tissue: HepG2 cells
cell type: hepatocarcinoma
treatment: None
chip-antibody: Santa Cruz mouse monoclonal sc7292x
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| Treatment protocol |
HepG2 cells were either cultured as above under proliferating conditions, or cultured with 10 µM CsA (Sigma-Aldrich) for 72 h before harvesting for analyses.
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| Growth protocol |
HepG2 cells were cultured in DMEM/F12 with 10% fetal calf serum and 1% penicillin-streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
ChIP-seq reads were aligned to the hg19 reference genome using bowtie2 version 2.25.0
Duplicate reads were removed using picard MarkDuplicates
ChIP and Input experiments were ensured to have equal read depth for each chromosom individually by downsampling the deeper sequenced experiment using home built python script DownsampleSam
Peak calling was done using EDD version 1.1.15, run 10 times on each samples in order to pptimized binSize and GapPenalty settings
LAD with a length less than the estimated Edd technical replicat resolution were reomove (19 on 951 total LADs, or <2 % of domains )
processed data files format and content: bed files with 3 columns: chromosome, start position, end position, score
processed data files format and content: bedgraph files with 4 columns: chromosome, start position, end position, score
Genome_build: hg19
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| Submission date |
Sep 07, 2018 |
| Last update date |
Feb 19, 2019 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
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| Department |
Institute of Basic Medical Sciences
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| Street address |
PO Box 1112 Blindern
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| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE119631 |
Characterization of the dynamics of lamin A and lamin B LADs in HepG2 cells: impact of cyclosporin A |
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| Relations |
| BioSample |
SAMN09989368 |
| SRA |
SRX4654318 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3379696_HepG2_ChIPSeq_Ctrl_LMNAC_log_ratio.bedgraph.gz |
2.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM3379696_HepG2_ChIPSeq_Ctrl_LMNAC_peaks.bed.gz |
2.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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