|
| Status |
Public on Aug 12, 2010 |
| Title |
ChIP-Seq for Med1 in mES cells (rep 2) |
| Sample type |
SRA |
| |
|
| Source name |
Embyonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
chip antibody: Med1/TRAP220
antibody catalog number: Bethyl A300-793A
antibody lot number: A300-793A-1
strain: C57BL/6-129
cell type: V6.5 embryonic stem cells
|
| Growth protocol |
V6.5 (C57BL/6-129) murine ES cells were grown under typical ES conditions on irradiated mouse embryonic fibroblasts (MEFs). For location analysis, cells were grown for two passages off of MEFs, on gelatinized tissue-culture plates, with the exception of replicate 1 of Med12, which was grown for one passage off of MEFs.
Mouse embryonic fibroblasts were grown in DMEM with 10% FBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies. Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 350bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against Med1
|
| Data processing |
Images acquired from the Illumina/Solexa sequencer were processed through the bundled Solexa image extraction pipeline which identified polony positions, performed base-calling and generated QC statistics.
Sequences were aligned using ELAND software to NCBI Build 36 (UCSC mm8) of the mouse genome. Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. The .ylf files are in [Y]oung [L]ab [F]ormat. It is a compact flat file representation of aligned read locations. The file contains the locations of the ELAND aligned reads with the following format: #U0 (read category U0) >1 (chromomsome 1) -12345 (minus strand read) -9876 (minus strand read) 54321 (plus strand read) >2 (chromomsome 2) -67890 (minus strand read) 98765 (plus strand read) 132323 (plus strand read) #U1 (read category U1) (etc.)
Each read was extended 200bp, towards the interior of the sequenced fragment, based on the strand of the alignment. Across the genome, in 25 bp bins, the number of ChIP-Seq reads within each bin was tabulated (replicates combined). Bins with at least 0.5 reads per million were visualized in the WIG files. The 25bp genomic bins that contained statistically significant ChIP-Seq enrichment were identified by comparison to a Poissonian background model, using an 1e-09 cutoff. Enriched bins within 200bp of one another were combined into regions. The Poissonian background model assumes a random distribution of background reads, however we have observed significant deviations from this expectation. Some of these non-random events can be detected as sites of apparent enrichment in negative control DNA samples and can create many false positives in ChIP-Seq experiments. To remove these regions, we compared genomic bins and regions that meet the statistical threshold for enrichment to a set of reads obtained from Solexa sequencing of DNA from whole cell extract(s) (WCE) in matched cell samples [GSM307154 and WCE sample uploaded here for mES, GSM555166 for MEFs]. We required that enriched bins and enriched regions have five-fold greater ChIP-Seq density in the specific IP sample, compared with the control sample, normalized to the total number of reads in each dataset. This served to filter out genomic regions that are biased to having a greater than expected background density of ChIP-Seq reads. For further details see associated publication.
|
| |
|
| Submission date |
Jun 25, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard A Young |
| E-mail(s) |
[email protected]
|
| Phone |
617-258-5219
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Young Lab
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (2) |
| GSE22557 |
Control of Embryonic Stem Cell State by Mediator and Cohesin |
| GSE22562 |
Control of Embryonic Stem Cell State by Mediator and Cohesin (Illumina ChIP-Seq data) |
|
| Relations |
| SRA |
SRX022695 |
| BioSample |
SAMN00016714 |
