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| Status |
Public on Sep 15, 2022 |
| Title |
ChIPseq_BT869_SMARCA4ko_H3K27me3 |
| Sample type |
SRA |
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| Source name |
BT869
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| Organism |
Homo sapiens |
| Characteristics |
cell line: BT869
cell type: H3.3K27M-glioma
genotype: SMARCA4 knockout
treatment: spiked-in with 200 ng of Drosophila chromatin
time: 4 days
chip antibody: H3K27me3, Active Motif, #39155
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| Growth protocol |
BT869 cells were grown as neurospheres in tumor stem media base supplemented with B27 minus vitamin A (Thermo Fisher Scientific), human growth factors (EGF, FGF, PDGF-AA, PDGF-BB [Shenandoah Biotechnology]) and heparin (Stemcell Technologies) in ultra-low attachment flasks, humidified atmosphere, 5% CO2, and at 37C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets were frozen at -80C, and sent to Active Motif Services (Carlsbad, CA) to be processed for ChIPseq. Cells were fixed with 1% formaldehyde for 15 min and chromatin was sheared sheared to an average length of 300-500 bp.
Illumina sequencing libraries were prepared from the ChIP and input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. Steps were performed on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina’s NextSeq 500 (75 nt reads, single end).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads were aligned using the BWA algorithm (default settings). Duplicate reads were removed and only uniquely mapped reads were used.
Human reads were normalized to Drosophila reads and extended in silico at their 3’-ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files and genome tracks were visualized by loading such files into IGV.
H3K27ac and BRG1 peak locations were determined using the MACS algorithm (v2.1.0) with a cutoff of p-value = 1e-7. H3K27me3-enriched regions were identified using the SICER algorithm at a cutoff of FDR 1E-10 and a max gap parameter of 600 bp.
Peaks that were on the ENCODE blacklist of known false ChIPseq peaks were removed.
Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations.
Assembly: hg38 and dm3
Supplementary files format and content: bigwig
Supplementary files format and content: excel file with the locations of BRG1 peaks
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| Submission date |
Sep 05, 2022 |
| Last update date |
Sep 15, 2022 |
| Contact name |
Joana Graca Marques |
| E-mail(s) |
joanagracamarques@gmail.com
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| Phone |
8572506823
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| Organization name |
Dana-Farber Cancer Institute
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| Street address |
360 Longwood Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE212700 |
BAF complex maintains glioma stem cells in pediatric H3K27M-glioma [ChIP-seq] |
| GSE212786 |
BAF complex maintains glioma stem cells in pediatric H3K27M-glioma |
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| Relations |
| BioSample |
SAMN30674684 |
| SRA |
SRX17428240 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6543710_4_0AB6_01ESDFCI_BCH869-SMARCA4-KO_H3K27me3_hg38_i50_dmnorm_signal.bw |
70.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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