|
| Status |
Public on May 06, 2015 |
| Title |
EpiSCs-H3K27me3 |
| Sample type |
SRA |
| |
|
| Source name |
Epiblast stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: E5.75 derived EpiSCs
passages: 20-25
strain: OCT4-deltaPE-GFP x C57BL/6
chip antibody: H3K27me3 (Millipore 07-449, lot# 2318778)
|
| Growth protocol |
Mouse EpiSCs were grown on MEFs in N2B27 media containing 12ng/ml FGF2 and 20ng/ml Activin-A supplementated with 20% KSR; rsEpiSCs were grown on MEFs in N2B27 media containing 20ng/ml and IWR1 2.5uM; Human H1 ESCs were grown on Matrigel-coated plates in mTeSR1 media, Human H1 rsESCs were grown on Matrigel-coated plates in customized mTeSR1 media containing 20ng/ml FGF2 and 2.5uM IWR1.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde at 37C for 10min, quenched with glycine at 37C for 5 min. Fixed cells were sonicated using Epishear (Active Motif) to achieve 200-700 size chromatin fragments. Solubilized chromatin was immunoprecipiated with antibody against H3K4me3 (Abcam 8580) and H3K27me3 (Millipore 07-449). Antibody-chromatin complexes were pulled down using Dynabeads protein A (Invitrogen), washed and then eluted. After cross-linking reversal, RNase and proteinase K treatment, immunoprecipiated DNA was purified using AMPure beads (Beckman Coulter).
ChIP DNA were end-repaired and 5' phosphorylated using T4 DNA Polymerase, Klenow and T4 Polynucleotide Kinase (Enzymatics). A single Adenine was added to 3' ends by Klenow (3-->5' exo-), and double-stranded Bioo Illumina Adapters (Bioo Scientific) were ligated to the ends of the ChIP fragments. Adapter-ligated ChIP DNA fragments were subjected to 15 cycles of PCR amplification using Q5 polymerase (NEB). AMPure beads were used to purify DNA after each step (Beckman Coulter). Pooled libraries were sequenced on the NextSeq 500 for single-end 75bp using high-output flowcell or on the HiSeq 2000 for single-end 36bp according to manufacturer’s instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIP-Seq for H3K27me3 in Epiblast stem cells
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2.
ChIP-Seq reads were aligned to the mouse (mm9) and human (hg19) genomes using bowtie2 (v2.1.0), keeping only reads that mapped to a unique genomic location (MAPQ > 10).
HOMER (v4.6) was used to process alignment files to generate normalized bedGraphs and bigWigs for the UCSC Genome Browser and quantify H3K4me3 and H3K27me3 levels at promoters.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: bigWig format. Initial reads were extended to approximately 150 bp and normalized coverage bedGraphs were generated using HOMER. The bedGraphToBigWig utility from the UCSC Genome Browser was then used to generate the bigWig files.
|
| |
|
| Submission date |
Nov 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE60604 |
An alternative pluripotent state confers interspecies chimaeric competency [ChIP-seq] |
| GSE60605 |
An alternative pluripotent state confers interspecies chimaeric competency |
|
| Relations |
| BioSample |
SAMN03216765 |
| SRA |
SRX835944 |
| Named Annotation |
GSM1552405_EpiSC-K27me3.ucsc.bigWig |
