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| Status |
Public on Dec 13, 2023 |
| Title |
Human male naive hESC WIN9 - H3K9ME3 |
| Sample type |
SRA |
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| Source name |
WIN1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Naive hESC
library strategy: ChIP-seq
experiment type: H3K9me3 antibody
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| Treatment protocol |
In addition to growing each cell type in appropriate media condition, cells were not treated with any additional agent(s).
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| Growth protocol |
Female naïve iPSCs and WIN1 male naïve hESCs used in ChIP-seq experiment were cultured in 5iLAF media in conditions previously described (Theunissen et al., 2014). 5iLAF media consisted of a 1:1 mixture of DMEM/F12 and Neurobasal (Life Technologies), supplemented with 1x N2 (Life Technologies), 1x B27 (Life Technologies), 1x penicillin/streptomycin, 1x nonessential amino acids, 0.5x GlutaMAX, 0.5% KSR, 0.1mM βmercaptoethanol, 50µg/ml bovine serum albumin (Sigma), 20ng/ml rhLIF (EMD Millipore), 20ng/ml Activin A (Peprotech), 8ng/ml FGF2, 1µM MEK inhibitor PD0325901 (Stemgent or Bio-Techne), 0.5µM B-Raf inhibitor SB590885 (Bio-Techne), 1µM GSK3β inhibitor IM-12 (Enzo), 1µM Src inhibitor WH-4- 023 (A Chemtek), and 10µM ROCK inhibitor Y-27632. For passaging, cells were dissociated by a 3 min treatment with StemPro Accutase (Life Technologies) at 37°C and re-plated after passing through a 40µm cell strainer in 5iLAF medium. Naïve hESCs were passaged as single cells every 5−6 days. In general naïve hPSCs were grown on irradiated DR4 mouse embryonic fibroblast feeder cells and maintained in a humidified 37°C incubator at 5% CO2 and atmospheric oxygen levels. Prior to ChIP experiments, a feeder depletion step was introduced by allowing feeder cells to attach to plastic surfaces for 40 min at 37oC and collecting only the non-attached PSC fraction.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone Modifications were assesed by means of X-linked ChIP
Histone modification genome-wide position data generated in this study were acquired using ChIP after crosslinking cells. Briefly, cells were grown to a final concentration of 5x107 cells for each ChIP-seq experiment. Cells were chemically cross-linked at room temperature by the addition of formaldehyde to 1% final concentration for 10 minutes and quenched with 0.125 M final concentration glycine. Cross-linked cells were re-suspended in sonication buffer (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS) and sonicated using a Diagenode Bioruptor for three 10-minute rounds using pulsing settings (30 sec ON; 1 min OFF). 10 ug of sonicated chromatin was then incubated overnight at 4oC with 5 ug of antibodies-conjugated to magnetic beads. Following the IP the beads were washed twice with RIPA buffer (50mM Tris-HCl pH8, 150 mM NaCl, 2mM EDTA, 1% NP-40, 0.1% Na-deocycholate, 0.1% SDS), low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10mM Tris pH 8.1, 250mM LiCl, 1mM EDTA, 1% Na-deoxycholate, 1% NP-40), and 1xTE. Finally, DNA was extracted by reverse crosslinking at 60oC overnight with proteinase K (20ug/ul) and 1% SDS followed by phenol:chloroform:iso-amylacohol purification. Libraries were constructed as indicated below and sequenced using paired-end 50 bp sequencing reactions
TruSeq illumina barcodes were ligated to immunoprecipitated DNA following end-repair. Minimal PCR amplification of 7-9 cycles produced suffienct material for sequening on a NovaSeq S2.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ChIP-seq using H3K9me3 antibody in female navie hESCs (WIN1)
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| Data processing |
Align: Bowtie2 (v2.2.9) with the default parameters was used to align reads
Filter: Reads that aligned to a unique position with no more than two sequence mismatches were retained. Multiple reads mapping to the exact same location and strand in the genome were collapsed to a single read to account for clonal amplification effects.
Count: Bedtools (v2.26.0) intersect was used to count reads in each genomic region (100kb windows every 25kb)
Enrichment: read counts in each window were normalized by the total read counts in each sample. The ratio of the normalized reads in the pulldown to the input were used as inputs
Assembly: hg38
Supplementary files format and content: bedgraph files represent normalized enrichment ratios in windows across the genome (100kb windows every 25kb). Is_unmappable=yes mark unmappable genomic regions
Supplementary files format and content: bedgraph files with chromatin states
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| Submission date |
Aug 22, 2023 |
| Last update date |
Dec 13, 2023 |
| Contact name |
Kathrin Plath |
| Organization name |
UCLA
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| Street address |
BOX 951737, 36-133 CHS
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-1737 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE241442 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells [ChIP-seq] |
| GSE241444 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells |
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| Relations |
| BioSample |
SAMN37116481 |
| SRA |
SRX21457862 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7729061_ChIPseq_WIN1hESC_H3K9ME3_100kb_25kb.bedgraph.gz |
686.7 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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