|
| Status |
Public on Jun 25, 2015 |
| Title |
ChIP-seq, RNAPII_HighDensity_noDMI |
| Sample type |
SRA |
| |
|
| Source name |
hMSC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Mesenchymal Stem Cells
chip antibody: RNAPII, sc-899, sc-9001, Santa Cruz
|
| Treatment protocol |
hMSCs were treated with DMI adipogenic induction medium for 24 hours (RNAPII) or 6 hrs (CEBPb, GR).
|
| Growth protocol |
hMSCs were cultured in low glucose DMEM supplemented with 1% glutamine, and 10% FBS. For cell seeding density experiments, low cell density refers to 3000 cells/cm2, and high density to 35000 cells/cm2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets were prepared from 1.8x106 cells, crosslinked in 1 % formaldehyde for 15 minutes. A 5 minute quench was performed with 125mM glycine. Cells were harvested from 10 or 15cm dishes in DPBS using a cell scraper. To prepare ChIP extracts, nuclei were suspended SDS lysis buffer (50 mM HEPES/NaOH pH 7.5, 1% SDS, 10 mM EDTA, Complete protease inhibitor), incubated at 4 C for 10 min, and subjected to microtip probe sonication under conditions optimized for IP efficiency.
ChIP-Seq libraries were produced and sequenced according to Illumina protocols. Libraries were multiplexed for sequencing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
MSC_HDD0_RNAPII
|
| Data processing |
Reads were mapped to the hg19 genome using Bowtie. Ambiguous alignments were excluded for TF ChIP-seq tracks. For H3K27Ac, reads were included with alignment locations of up to 3 possible loci.
Redundant reads were removed from TF ChIP-seq datasets using the makeTagDirectory script in HOMER. Redundant reads were preserved only for the H3K27Ac dataset.
Bed files for TF ChIP-Seq peaks were generated using the FindPeaks script in HOMER.
Bedgraph file for H3K27Ac was generated using the makeUCSCfile script in HOMER.
RNAPII.peaks.bed file was produced using a slope-based feature detection algorithm in Galaxy, see Cohen et al. ELife for details.
Input bias regions were manually curated from genomic regions showing strong ChIP enrichment on the genome browser independently of ChIP antibody.
Genome_build: hg19
Supplementary_files_format_and_content: 1 )TF ChIP-seq peak files in BED format 2) H3K27Ac in BEDGRAPH format
|
| |
|
| Submission date |
May 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
David J Steger |
| E-mail(s) |
stegerdj@pennmedicine.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Institute of Diabetes, Obesity, and Metabolism
|
| Lab |
Steger
|
| Street address |
12-193 SCTR 3400 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE68864 |
ATF4 Licenses C/EBPb Activity in Human Mesenchymal Stem Cells Primed for Adipogenesis |
|
| Relations |
| BioSample |
SAMN03657562 |
| SRA |
SRX1027434 |
