|
| Status |
Public on Jan 01, 2010 |
| Title |
MTF2_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic Stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129SvJae x C57BL/6
cell type: embryonic stem cells
passages: 10-15
chip antibody: MTF2
|
| Growth protocol |
ES cell was cultured in 15% FCS/DMEM, supplemented with Pen/Strep, Glutamax, MEM non-essential amino acids, ESGRO and 2-mercaptoethanol. ES cells were passaged off feeder cells for ~2-3 passages before harvesting for ChIP experiments.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired and 5' phosphorylated using END-It DNA End Repair Kit (Epicenter). Then, DNA was treated with Klenow fragment (3' to 5', exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp to 70bp (insert plus adaptor and PCR primer sequences) were isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP against MTF2
|
| Data processing |
For BED files: reads (of 36bps) produced by the Illumina platform were aligned by MAQ. Sequences with more than a single best matche in the reference genome were discarded. We also created a map of ?unalignable? genomic positions to which no unique 36 base read could be uniquely aligned due inherent sequence redundancy for consideration in downstream processing.
For WIG files: The short sequence reads correspond to the end of the DNA fragments in the library. Since the average size of the ChIP fragments is in the range of 200 bp, we extended the aligned reads in silico to a total length of 200 bases. The entire genome was divided to non-overlapping windows of 25bps. The signal in a given window was calculated as the number of extended reads that it fall within that window.
|
| |
|
| Submission date |
Dec 30, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Manching Ku |
| E-mail(s) |
MKU@mgh.harvard.edu
|
| Organization name |
MGH
|
| Street address |
185 Cambridge St. CPZN-8400
|
| City |
boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE19708 |
Jarid2 and PRC2, Partner in Regulating Gene Expression |
|
| Relations |
| SRA |
SRX015777 |
| BioSample |
SAMN00007512 |
