|
| Status |
Public on May 01, 2017 |
| Title |
EpiSCA_SOX2_ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
Epiblast stem cells_freed from Activin
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129/Sv
chip target: SOX2
chip reagent: Streptavidine
tag: biotin
|
| Treatment protocol |
A suspension of EpiSCs following Accutase treatment was plated on a 10 cm diameter dish and immediately transfected with 5 mg of pCAGGS-BLRP-BirA plasmid to express BLRP-tagged plasmid and Lipofectamine 2000 (Thermo Fisher). After 8 h, the culture medium was refreshed and 5 mM biotin was added.
|
| Growth protocol |
Feeder-free epiblast stem cell culture in N2/B27-supplemented DMEM/F12 with 20 ng/ml activin and 10 ng/ml Fgf2. EpiSCA cultures were freed from activin for 18 hr before harvest, without significant changes in microarray data and called ChIP-seq peaks.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After 24 h, the cultures were fixed with 1% formalin for 10 min at room temperature, and then the cells were scraped from dishes, washed, collected in a tube and lysed using the lysis buffer containing 1% SDS. Cell lysates were sonicated in a 1–ml vial using COVARIS M220, which produced an average DNA fragment size of approximately 150 bp. The lysate was mixed with streptavidin beads overnight and washed with a series of solutions described in Matsuda K et al. (submitted).
The DNA fragments were ligated with adapters and amplified by PCR using TruSeq ChIP Sample Prep Kit (Illumina), according to the manufacturer’s directions, except that we terminated the PCR reaction at 10 cycles.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie2.
ChIP-seq peaks were called using MACS1.4, input DNA without ChIP as reference, and the default settings except for band width, which was adjusted to 150 bases.
Genome_build: mm9
Supplementary_files_format_and_content: Bigwig files were generated using SraTailor
|
| |
|
| Submission date |
Nov 03, 2015 |
| Last update date |
Aug 27, 2019 |
| Contact name |
Hisato Kondoh |
| E-mail(s) |
kondohh@cc.kyoto-su.ac.jp
|
| Phone |
81-72-681-9762
|
| Organization name |
Biohistory Research Hall
|
| Street address |
Murasakicho 1-1
|
| City |
Takatsuki |
| State/province |
Osaka |
| ZIP/Postal code |
569-1125 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE74636 |
Genome-wide maps of five major transcription factors in epiblast stem cells |
|
| Relations |
| BioSample |
SAMN04231643 |
| SRA |
SRX1410926 |
