 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 24, 2013 |
| Title |
10kCells-ES-input-WGA |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cell
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Embryonic stem cell
number of cells: 10k Cells
strain: E14
ChIP: none, input-DNA
|
| Treatment protocol |
Gonads were treated with 0.25% Trypsin-EDTA (Gibco) for 10-15 min at 37 C, and trypsin was inactivated by addition of 15% Fetal Bovine Serum (FBS)/ DMEM (Gibco). Cells were filtered through 40 µm cell strainers (BD Falcon) to obtain single cell suspension for FACS sorting (FACSAria II SORP, BD Bioscience).
|
| Growth protocol |
Gonads were microscopically dissected from embryonic day 11.5 and 13.5 embryos, which were derived from the cross between male homozygous Pou5f1-GFP mice (Jackson’s lab, stock no. 004654) and female wild type CD1 mice.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed as described previously (Loh et al., 2006). Cells were crosslinked with 1% formaldehyde for 8 min at room temperature and the formaldehyde was quenched with 125 mM glycine. FACS-purified cells were fixed, quenched and rinsed with TBSE buffer twice before storage at -80 degree Celsius until sufficient cell numbers are accumulated for ChIP experiments. Cell lysates were sonicated using the bioruptor (Diagenode) and chromatin extracts were immunoprecipitated with anti-H3K4me3 (04-745, Millipore), anti-H3K27me3 (07-449, Millipore), anti-H3K27ac (ab4729, Abcam) and H2BK20ac (ab52988, Abcam) antibodies. Following de-crosslinking, small-scale ChIP samples were subjected to an additional 15 cycles of amplification using the GenomePlex Single Cell Whole Genome Amplification Kit (WGA4, Sigma) and further purified using the QIAquick PCR purification kit. For all ChIP experiments, quantitative PCR analyses were performed in real-time using the ABI PRISM 7900 Sequence Detection System and SYBR Green Master Mix. Relative occupancy values were determined by calculating the immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample). For ChIP-Seq library preparation (ChIP-Seq DNA Sample Prep Kit IP-102-1001, Illumina), 5-15 ng of ChIP DNA was used for end-repair and adaptor ligation followed by 15 cycles of amplification. 200-300 bp size fragments were selectively cut from the agarose gel and purified using the Qiagen gel extraction kit. Small-scale ChIP samples were subjected to additional processing steps to excise PCR adaptors prior to ChIP-Seq library preparation. The extracted DNA was quantified using Picogreen assay and subjected to ChIP-Seq sequencing for 36 cycles according to the manufacturer’s instructions (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
wiggle track without GC bias correction
|
| Data processing |
All the ChIP-Seq and input control tag data were binned in form of 200 bp bins. GC bias correction was done for ChIP-Seq data using respective input controls. Peak detection was done on binned GC-corrected signal after convolving it with a kernel just as in F-Seq, however here, kernels were first learned using enriched regions for each ChIP-Seq data. ChIP-Seq peaks overlapping repeats (RNA repeats, Satellites and segmental duplications) and peaks in input control were removed as false positives.
Genome_build: mm8
|
| |
|
| Submission date |
Sep 11, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Vibhor Kumar |
| E-mail(s) |
kumarv1@gis.a-star.edu.sg
|
| Organization name |
Genome Institute of Singapore
|
| Department |
CMB-6
|
| Street address |
60 Biopolis Street, Genome, #02-01
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9185 |
| Series (2) |
| GSE38164 |
In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures [ChIP-Seq] |
| GSE38165 |
In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures |
|
| Relations |
| SRA |
SRX186068 |
| BioSample |
SAMN01164159 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1001324_trackf-Input-10k.vstep.wig.gz |
24.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
