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| Status |
Public on Dec 01, 2017 |
| Title |
HR2 AS DNM3BKO iPS Input |
| Sample type |
SRA |
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| Source name |
HR2 AS DNM3BKO iPS Input
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| Organism |
Homo sapiens |
| Characteristics |
source cell type: B-lymphocyte
cell type: iPSC derived from Angelman syndrome patient
genotype/variation: DNM3B gene was knocked out
genotype/variation: CpG free neo cassette is integrated in SNURF/SNRPN locus
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| Biomaterial provider |
Coriell Cell repositories; http://ccr.coriell.org/
http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM20409
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| Treatment protocol |
Cells were fixed with 1% formaldehyde at 37 °C for 10 min and then quenched with glycine at 37 °C for 5 min.
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| Growth protocol |
ESCs and iPSCs were cultured with TeSR medium on a Matrigel coated plate.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were sonicated using Bioruptor to achieve 200–700 bp size chromatin fragments. Solubilized chromatin was immunoprecipiated with antibody against H3K4me3 (Abcam 8580). 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. Adaptor-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 HiSeq2500 (V4 Chemistry) for single-end 50 bp according to the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequenced reads were quality-tested using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and aligned to the hg19 (Genome Sequencing Consortium 2001) human genome using the STAR aligner (Dobin 2013) version 2.4.0k. Mapping was carried out using default parameters (up to 10 mismatches per read, and up to 9 multi-mapping locations per read).
The genome index was constructed using the gene annotation supplied with the hg19 Illumina iGenomes collection (http://support.illumina.com/sequencing/sequencing_software/igenome.html) and sjdbOverhang value of 100.
Reads uniquely mapping to the genome (>80%) were then used for peak calling with Homer (Heinz 2010), assuming a peak size of 500, extending the peaks to cover the full enriched region, assuming a fold enrichment of at least 4 over input reads, a poisson p-value threshold relative to input count of 1e-4, and at least 25 normalized read counts per peak.
Peaks from WTiPS, ASiPS, and ASHR2neoex samples, or peaks from ASDM3BKO, HR4ASDM3BKO, and HR14ASDM3BKO were merged using the Homer mergePeaks.pl program, and normalized read counts in peaks were used to calculate log fold change between conditions.
Homer makeUCSCfile.pl was used to generate normalized read count density tracks for peak visualization.
genome build: hg19
processed data files format and content: Gzipped bedGraph tracks show a continuous normalized (total of 1e7 reads) read alignment of reads to the hg19 genome. These files can be loaded into a genome browser to visualize and compare ChIP peaks. Input controls are indicated with _Input.
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| Submission date |
Dec 13, 2016 |
| Last update date |
Mar 28, 2022 |
| Contact name |
April Elizabeth Williams |
| E-mail(s) |
apriljack06@gmail.com, awilliams@salk.edu
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| Phone |
7345461645
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| Organization name |
Salk Institute for Biological Studies
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| Department |
IGC
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| Street address |
10010 N Torrey Pines Rd
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| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE63938 |
Targeted CpG island methylation in human pluripotent stem cells |
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| Relations |
| BioSample |
SAMN06140245 |
| SRA |
SRX2420180 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2427970_1_HR14ASDM3BKO_Input_S42.ucsc.bedGraph.gz |
180.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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