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| Status |
Public on Jul 20, 2014 |
| Title |
K27 GSKJ4 24h, rep2 |
| Sample type |
SRA |
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| Source name |
Pediatric, K27M mutated DIPG cells treated with GSKJ4 for 24h, H3K27me3 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SF8628
age: year 3
tissue: pons
gender: female
treatment: GSKJ4
treatment duration: 24h
chip antibody: anti-H3K27me3
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| Treatment protocol |
Cells were treated with vehicle or 6 μM GSKJ4 for 24 and 72 hours.
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| Growth protocol |
Primary pediatric human glioma cells, designated with “SF”, were obtained from surgical biopsy of tumor from patients admitted to UCSF Medical Center, and in accord with an institutionally approved protocol. Establishment of glioma cell cultures, from surgical specimens, and tumor cell modification for expression of firefly luciferase, for in vivo bioluminescence imaging, have been described in Hashizume R et al., 2012 (PMID 22983601).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 1×10^6 cells, treated with vehicle or 6 μM GSKJ4 for 24 and 72 hours, were harvested, washed with PBS and fixed with 1% formaldehyde for 5 minutes at room temperature. Cells were then quenched with 125 mM glycine for 5 minutes. After wash, cell extracts were prepared using lysis buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-deoxycholate, 1 mM PMSF, 1 mM Pefoblock, 1 mM benzamidine, 1 mg/ml bacitracin) on ice for 10 minutes.
Chromatin was sheared by sonication (bioruptor, High power, 15 × 2 cycles), to average lengths of 500 bp, then immunoprecipitated using an antibody against histone H3K27me3 (#9733, Cell Signaling). After repeated washings, the DNA was recovered from the beads by incubating the beads in elution buffer (10 mM Tris at pH 8.0, 10 mM EDTA at pH 8.0, 1% SDS, 150 mM NaCl, 5 mM DTT) at 65 oC. Both input DNA and eluted DNA were subsequently purified using Qiagen Mini Elute PCR purification kit. ChIP DNA libraries were prepared with the Ovation Ultralow DR Multiplex system (NuGEN).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
8628_K27_24h_6uM
DIPG-derived cell line, harboring H3F3A K27M mutation.
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| Data processing |
ChIP-seq reads were aligned to the human genome (hg19) using Bowtie2 software and preset parameters. Only uniquely mapping reads were used for further analysis.
Informative tags were selected from the aligned ChIP-seq data of each sequenced sample based on the cross-correlation profile of positive- and negative-strand tag densities. Background anomalies resulting from extremely high-density peaks at single chromosome positions were removed. After these filtering steps, the remaining tag list from each K27me3 immunoprecipitated sample was normalized against tag lists from sample matched input DNA (treated only with DMSO). Broad regions of binding enrichment (i.e. ChIP signal over input) for each immunoprecipitated sample were obtained by comparing scaled ChIP and input tag counts to see if their ratio exceeds that expected from a Poisson process. The R library SPP was used to perform all aforementioned steps.
Distance to the nearest transcription start site (TSS) was obtained for each enriched binding region using Bioconductor package ChIPpeakAnno.
Genome_build: GRCh37
Supplementary_files_format_and_content: bed files were generated using R library SPP.
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| Submission date |
Jul 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Rintaro Hashizume |
| E-mail(s) |
rintaro.hashizume@ucsf.edu
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| Organization name |
UCSF
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| Department |
Neurosurgery
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| Street address |
1450 3rd street
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| City |
San Francisco |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE59496 |
Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma [ChIP-seq] |
| GSE59497 |
Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma |
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| Relations |
| BioSample |
SAMN02919173 |
| SRA |
SRX655399 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1438426_8628_K27_24h_6uM.FCC3Y8MACXX_L8_IGGCTAC.bed.gz |
2.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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