|
| Status |
Public on Aug 30, 2017 |
| Title |
MRC5_rep2_PolII |
| Sample type |
SRA |
| |
|
| Source name |
MRC5_PolII
|
| Organism |
Homo sapiens |
| Characteristics |
chip antibody: Rpb1 CTD (Cell Signaling 2629S Lot: 3)
cell line: MRC5 fibroblasts
passage: 8
|
| Treatment protocol |
About 7-8 x 105 MRC5 fibroblasts were seeded on 10 cm plates on day 0. These cells were grown in EMEM (ATCC) media supplemented with 10% FBS. Twenty-four hours later, two plates were trypsinzed to get an estimate of average number of cells in each 10 cm plate. Cells were then infected with lentiviruses separately encoding either OSKM or OS(v)KM at multiplicities of infections (MOIs) that were determined to be optimal for reprogramming (see Suppl Table 2). Media was changed daily until the time of harvest on day 5. At the time of harvest, cells on plates were washed once with PBS (room temperature) and cross-linked by adding EMEM + 10% FBS containing 1% fomaldehyde. Plates were returned to the incubator and incubated for 10-15 minutes. Cells were washed twice with ice cold PBS containing a cocktail of protease inhibitors (Sigma Cat # P8340; 10ml/ml PBS) and 1mM phenylmethylsulfonyl fluoride (PMSF). Cross-linked cells from each condition (Mock infected MRC5, OSKM or OS(v)KM; all at passage 8) were scraped using a cell scraper, pooled in 15 ml tubes and pelleted. We typically pooled cross-linked cells from at least 4-5, 10cm plates per condition (~ 5 million cells) for each ChIP-Seq assay. As controls, we harvested approximately 5 million cross-linked human ES (H9 hESCs, p. 35) or human iPS cells (an iPSC line derived using OS(v)KM (3S2 hiPSC, p.20) from on 6 cm plates and grown in feeder-free conditions (on vitronectin-coated plates and grown in Essential 8 media).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
See ChIP-Endo Protocol.pdf
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Sample 1705
[Processed data files]:
MRC5_avg_PolII_Bin_2000_117.bed.gz
MRC5_avg_PolII_Bin_2000_117.wig.gz
MRC5_rep2_PolII_Bin_2000_1705.bed.gz
MRC5_rep2_PolII_Bin_2000_1705.wig.gz
gene_MRC5_avg_PolII_117.bed.gz
gene_MRC5_avg_PolII_117.bedGraph.gz
gene_MRC5_rep2_PolII_1705.bed.gz
gene_MRC5_rep2_PolII_1705.bedGraph.gz
|
| Data processing |
cutadapt-1.9.1 cutadapt -a AGATCGGAAGAGC
bwa-0.7.13 bwa aln
bwa-0.7.13 bwa sampe
all duplicates eliminated
values at all features are calculated by (number of midpoints of tags / bp of feature) / (total number of unique tags / genome length)
above described values are quantile normalized
Genome_build: hg19
Supplementary_files_format_and_content: bed, bedGraph, and wig.
|
| |
|
| Submission date |
Jun 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark Ptashne |
| E-mail(s) |
m-ptashne@mskcc.org
|
| Organization name |
Sloan Kettering Institute
|
| Department |
Molecular Biology Program
|
| Lab |
Mark Ptashne
|
| Street address |
1275 York Ave
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE81899 |
OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts |
| GSE81900 |
OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts |
|
| Relations |
| BioSample |
SAMN05194831 |
| SRA |
SRX1813537 |
