|
| Status |
Public on Aug 13, 2013 |
| Title |
QSC_y_input DNA |
| Sample type |
SRA |
| |
|
| Source name |
hindlimb muscles
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: male
age: 2-3 months
sorting markers: VCAM+ / CD31- / CD45- / Sca1-
chip antibody: none
|
| Treatment protocol |
FACS-purified QSCs from young mice were plated on ECM coated surface in F10 media supplemented with 10% horse serum for 3 days to obtain ASCs.
|
| Growth protocol |
QSCs were FACS-purified from hindlimb muscle of healthy mice at the indicated ages.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
FACS-sorted cells were crosslinked with 1% formaldehyde at room temperature with gentle agitation for 10 minutes. The crosslink was terminated by Glycine at a final concentration of 0.125 M. Cells were then washed three times with ice-cold PBS and stored at -80°C until a sufficient number of cells had been obtained. ChIP was performed following standard ChIP protocols with the following major modifications: 10E6 cells were resuspended in 200 ml lysis buffer and sonicated to obtain DNA fragments from 150 to 500 bp in size. Each ChIP reaction was performed with 10E6 cells and 5 ug of antibody.
The ChIP DNA was size-selected for fragments between 150-400 bp by 2% low-melt agarose gel electrophoresis (NuSieve). A library for deep sequencing was generated with Illumina ChIP-seq Sample Prep Kit (Part# IP-102-1001) with the following modification to the standard protocol: The adaptor solution was diluted 50 times for ligation. 15 cycles of PCR amplification were performed immediately following adaptor ligation. Size-selection of fragments between 200-400 bp was performed after PCR amplification. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer IIx following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalling was performed using Illumina RTA 1.8.70.0
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie.
Unique reads mapped to a single genomic location (allowing a maximal of 2 mismatches) were used for peak detection by Model-based Analysis of ChIP-Seq (MACS) with alignment of the ChIP input DNA as control.
Genome_build: mm9
Supplementary_files_format_and_content: .bed files for ChIP-seq analysis containing peak location and −10*log10(p value) are provided.
|
| |
|
| Submission date |
May 24, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ling Liu |
| E-mail(s) |
lingliu@stanford.edu
|
| Organization name |
Stanford University
|
| Street address |
300 Pasteur Drive
|
| City |
Palo Alto |
| ZIP/Postal code |
94306 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE47362 |
Epigenetic determinants of muscle stem cell quiescence and chronological aging (ChIP-seq) |
|
| Relations |
| BioSample |
SAMN02178766 |
| SRA |
SRX286491 |
