|
| Status |
Public on Apr 25, 2014 |
| Title |
ADF04DF_LMNA_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
normal primary dermal fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: --
cell type: normal primary dermal fibroblasts
passages: 5-7
chip-antibody: Santa Cruz sc-7292
|
| Biomaterial provider |
Norwegian Stem Cell Center
|
| Treatment protocol |
Cells were cultured as described under Growth protocol. Cells were cultured to confluency before harvesting to ensure consistency of cell cycle stages between the two cell types.
|
| Growth protocol |
Human normal dermal fibroblasts (Lonza CC-2511; LDFs) and human normal primary dermal fibroblasts (Norwegian Stem Cell Center AD04DFs) were cultured in DMEM/F12 containing 13% FCS, 2 ng/ml basic fibroblast growth factor and antibiotics. Cells were exponentially growing and used at passage 5-7. AD04DFs were obtained with Norwegian Ethics Committee Approval REK2617A.
|
| Extracted molecule |
genomic DNA |
| Extraction 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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Processed file created with EDD using as input HSF_AD04_Input.fastq.gz in addition to the raw file in this line
|
| Data processing |
ChIP-seq reads were aligned to the hg19 reference genome using bowtie2 version 2.1.0
Duplicate reads were removed using picard MarkDuplicates
ChIP and Input experiments were ensured to have equal read depth by downsampling the deeper sequenced experiment using picard DownsampleSam
Peak calling was done using EDD version 0.9
Genome_build: HG19
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Jan 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
|
| Department |
Institute of Basic Medical Sciences
|
| Street address |
PO Box 1112 Blindern
|
| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE54332 |
EDD: a program for detection of wide genomic enrichment domains robust against local variations [ChIP-Seq] |
| GSE54334 |
EDD: a program for detection of wide genomic enrichment domains robust against local variations |
|
| Relations |
| BioSample |
SAMN02595202 |
| SRA |
SRX447388 |
