|
| Status |
Public on Sep 09, 2013 |
| Title |
Proliferating Histone 3 ChIP R2 |
| Sample type |
SRA |
| |
|
| Source name |
Proliferating Histone 3 ChIP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
cell type: primary human fibroblasts
cell type: proliferating cells
population doubling: 24
chip antibody: H3
chip antibody vendor: Abcam
chip antibody cat. #: ab1791
chip antibody lot #: GR26510-1
|
| Treatment protocol |
Senescence cells were maintained in dishes for 2 weeks to ensure growth termination. Senescence was determined by monitoring p16 upregulation, downregulation of cyclin genes, and by SA-beta galactosidase staining. Rad-inducible cells were created by retrovirally infecting normal IMR90 fibroblasts with pLNC-ER:Ras; ras was induced by delivering 4-hydroxytamoxifen (4-OHT) at a final concentration of 100 nM when changing media.
|
| Growth protocol |
IMR90 cells (obtained from Coriell Institute for Medical Research, Camden, NJ) were grown in standard tissue culture medium (DMEM with 10% FBS and 1% penicillin streptomycin) at 3% oxygen. For senescence studies, cells were replicatively passaged until growth ceased. For OIS studies, cells were grown in phenol red-free DMEM + 20% FBS, L-glut and Pen/Strep (with 500 ug/ml G418).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells in 10cm2 dishes were fixed in 1% formaldehyde for 10 minutes and fixation was quenched with addition of glycine to 125mM for an additional 5 minutes. Cells were harvested by scraping from plates, and washed twice in 1xPBS before storage at -80C. ChIP was performed as described in the Young lab protocol, except that extracts were sonicated twice for 9 minutes each round (30 seconds sonication with intermediate incubation of 30 seconds per round) using a Bioruptor (Diagenode). All ChIPs were performed using 500µg of extract 2µg of antibody per sample. 30µl of Protein G Dynabeads (Invitrogen 100.02D) were used per ChIP. For sequencing, 10ng ChIP DNA was used to make sequencing libraries using standard Illumina library single-end construction procedures. Sequencing was performed on either Illumina GAIIx or Hi-Seq platforms (36bp, single-end and 50 or 100bp, single or paired-end reads, respectively).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
DNA immunoprecipitated by H3 (Abcam ab1791)
|
| Data processing |
Galaxy was used to perform base-calls
Alignment was performed with bowtie v0.12.5 for replicative H3 and H3K4me3 (paired-end), for OIS H3 and H3K4me3 (single-end), for lamin B1 (single-end alignment of paired-end data), and for H3K27me3 (single-end).
The processed data files aren't just tag-aligned BED files or wiggle files. They are locations of differential signal based on pairwise comparison of two samples. The replicative H3K4me3 mesas come from comparing GSM897556 and GSM897560 (normalized to the signal in GSM897555 and GSM897559). Likewise, the replicative H3K27me3 canyons and mesas come from comparing GSM897557 and GSM897561 (normalized the same way). The lamin B1 is scanned solely for regions of enrichment.
In-house scripts were used to detect canyons, mesas, and LADs among aligned sequence tags; these are available upon request
Genome_build: hg18
Supplementary_files_format_and_content: BED files were generated using scripts that detect canyons, mesas, and LADs and define the boundaries of these loci
|
| |
|
| Submission date |
May 07, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
|
| Department |
Cell & Developmental Biology
|
| Lab |
Zaret Lab
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE36616 |
Lamin B1 depletion in senescent cells triggers large-Scale changes in gene expression and in the chromatin landscape[ChIP-seq] |
| GSE36641 |
Lamin B1 depletion in senescent cells leads to large-scale changes in the chromatin landscape |
|
| Relations |
| BioSample |
SAMN02141288 |
| SRA |
SRX275792 |
