|
| Status |
Public on Oct 20, 2010 |
| Title |
PRDM14_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
Embyonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: pluripotent and self-renewing cells
antibody: custom made PRDM14
|
| Treatment protocol |
No treatment was performed for ESCs used for ChIP-sequencing.
|
| Growth protocol |
The hESC line H1 (WA-01, passage 28) was used for this study. They were cultured feeder free on matri-gel (BD). Condition medium used for culturing human contained 20% KO serum replacement, 1 mM L-glutamine, 1% non-essential amino acids and 0.1 mM 2-mercaptoethanol and an additional 8ng/ml of basic fibroblast growth factor (Invitrogen) supplemented to the hESCs unconditioned medium. Medium was changed daily.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with PRDM14 antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 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 (insert plus adaptor and PCR primer sequences) were band 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 II |
| |
|
| Description |
Chromatin IP against PRDM14
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the human (hg18) genome using the Illumina Genome Analyzer II Pipeline. All uniquely mapped reads with two or fewer mismatches were retained.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) using processed data file. Control input DNA sequencing library was used as background for peak calling.
|
| |
|
| Submission date |
Jul 06, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuriy L Orlov |
| E-mail(s) |
[email protected]
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Computational Systems Biology
|
| Street address |
60 Biopolis Street
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE22767 |
Genome-wide mapping of PRDM14 binding sites in human embryonic stem cells |
|
| Relations |
| SRA |
SRX023135 |
| BioSample |
SAMN00016914 |
