|
| Status |
Public on Apr 10, 2017 |
| Title |
PADI_inhibition_H3K9me3_ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
cell line: E14
treatment: CI-Amidine treatment
chip-antibody: Ab8898(Abcam)
|
| Treatment protocol |
Cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Fixation was subsequently inactivated by the addition of 125mM glycine.
|
| Growth protocol |
Undifferentiated mouse ES cells E14 was cultured under feeder-free condition, were plated onto gelatin-coated dishes in Dulbecco's modified Eagle medium (DMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum (FBS; GIBCO), 0.055 mM -mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF (Millipore ESG1107) in incubator set at 37 ºC in 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were scraped off dishes and collected by centrifuge. Cross-linked chromatin complexes were isolated from the Nuclei lysis buffer and then sonicated to obtain fragments in a size range between 350-500bp. H3R26Cit antibody (ab19847) were incubated with solubilized DNA fragments overnight at 4°C. Protein A/G agarose beads (Pierce, Rockford, IL) were used to capture the antibody-chromatin complex followed by washes and elution with 1% SDS. The DNA was recovered by reversing the cross-links with 20 mM NaCl at 65°C overnight. DNA was purified by QIAquick PCR Purification Kit (Qiagen 28106) and dissolving in a final volume of 30ul per immunoprecipitation.
The Protocol of KAPA High-Throughput Library Preparation Kit (Cat. No. KK8234)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Paired-end ChIP-Seq data were aligned to the mm9 genome assembly using Bowtie2 version 2.1.0 with maximum fragment length = 1,000; single-end ChIP-Seq data were mapped using bowtie2 with default parameters
Data were filtered to remove PCR duplicates using samtools rmdup
Data were filtered to keep reads with mapping quality higher than 5.
Paired end data were filtered to keep reads mapped in proper pair.
Peaks were called using MACS14 (1.4.0beta) with default parameters. IgG data for peak calling: GSM881345.
genome build: mm9
processed data files format and content: peak files in BED format
|
| |
|
| Submission date |
Dec 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sheng Zhong |
| E-mail(s) |
zhonglabucsd@gmail.com
|
| Organization name |
University of California, San Diego
|
| Department |
Bioengineering
|
| Lab |
Dr. Sheng Zhong
|
| Street address |
9500 Gilman Drive MC 0412
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0412 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE45338 |
Role of SMARCAD1 and H3R26Cit in Maintenance of the Naïve Pluripotent State |
|
| Relations |
| BioSample |
SAMN06133334 |
| SRA |
SRX2416688 |
