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| Status |
Public on Oct 22, 2020 |
| Title |
BMI1_ChIP_SF8628_PTC028 |
| Sample type |
SRA |
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| Source name |
diffuse intrinsic pontine glioma
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SF8628
passage: 10-15
chip antibody: Bmi1 - clone D42B3 - Rabbit mAb #5856 -from Cell Signaling Technologies
growth protocol: DMEM-High glucose (Invitrogen), 10% fetal bovine serum (Atlanta Biologicals), 1% Non-Essential Amino Acids, 1% Antibiotic-Antimycotic
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| Treatment protocol |
The small-molecule inhibitor used in this study is PTC-028 (orb181369, Biorbyt). This drugs was all dissolved in DMSO to make stock concentrations to use in in vitro experiments.
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| Growth protocol |
See SAMPLES section
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The growth medium was aspirated from cells (70% confluence) and replaced with a 1% formaldehyde in 1X DPBS for 10 min at room temperature. The cross-linking was terminated by addition of glycine to a final concentration of 0.125 M and incubation for 5 min. The cells were washed twice with cold PBS and scraped in ChIP lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8.1) containing COmplete EDTA free protease inhibitors (Sigma). The cell lysates were sonicated with a Bioruptor Plus (Diagenode) for 25 cycles (30 seconds ON and 30 seconds OFF). The size of the sonicated DNA fragments was checked on an 1X TAE1 % agarose gel electrophoresis and ranged between 250 to 500 bp. After removing the debris via centrifugation, chromatin extracts were collected and protein concentration was determined by BCA protein assay reagents (Pierce). For immunoprecipitation, chromatin extracts with 1 mg of total protein for each sample were incubated with primary antibodies (dilution 1:50) overnight at 4ºC. Primary antibodies used are included in table under “Western blotting” below. After incubation with the primary antibody, 20 uL of pre-washed magnetic beads (Magna ChIP Protein A+G Magnetic Beads, Millipore Sigma) were added to each sample for 2 hours at 4ºC. Using a magnetic rack, the immunoprecipitates were washed successively with 1 ml of low salt buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA), high salt buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA), LiCl washing buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, , 1.0% NP40, 1.0% deoxycholate, 1 mM EDTA) and twice with TE buffer. The DNA-protein complexes were eluted with 300 μl of IP elution buffer (1% SDS, 0.1 M NaHCO3). The eluents were pooled together and the cross-links were reversed by adding NaCl (a final concentration 0.2 M) into the eluents and incubating them at 65 ºC overnight. The DNAs were recovered by proteinase K and RNase A digestion, followed by phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended in 50 μl of 10 mM Tris-HCl [pH 8.0]. ChIP-DNA was quantified using the Qubit dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific).
Libraries were prepared according to Nugen's instructions accompanying their Ovation Ultralow System V2 kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The quality of fastq files was accessed using FastQC
The Illumina adapters and low quality reads were filtered using BBDuk with the parameters for paired-end reads
The ChIP-seq reads were aligned to the hg38 genome assembly using bowtie2 (v. 2.3.2).
Samtools (v. 1.5) was used to convert the sam files into bam files, to filter out unmapped reads and to sort the bam files.
When the total number of reads per sample was inferior to 40M, the libraries were re-sequenced and the additional bam files generated from the second sequencing were merged with the bam files from the first sequencing.
The total number of reads used for peak calling was normalized using samtools view -hs (random extraction of a specified percentage of reads). The same number of reads was used for the RNA polymerase II ChIP-seq and for the inputs.
Samtools sort was used to sort the bam files after read extraction.
Bam files were indexed using samtools index.
Bedtools genomecov was used to create bedgraph files from the bam files.
Igvtools toTDF was used to create tdf files from the bedgraph files.
Peaks were called using macs2 (v. 2.1.1.20160309) with default parameters using python (v. 2.7.14).
Peak locations were further annotated using the clusterProfiler, ChIPseeker and ngs.plot.r R packages.
Genome_build: hg38
Supplementary_files_format_and_content: The bam files were generated with samtools.
Supplementary_files_format_and_content: The tdf files were generated with igvtools toTDF.
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| Submission date |
Nov 21, 2019 |
| Last update date |
Oct 22, 2020 |
| Contact name |
Etienne Danis |
| E-mail(s) |
etienne.danis@cuanschutz.edu
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| Organization name |
University of Colorado, Denver - Anschutz Medical Campus
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| Street address |
12800 E. 19th Ave
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL27644 |
| Series (1) |
| GSE140768 |
Senescence induced by BMI1 inhibition is a therapeutic vulnerability in H3K27M-mutant DIPG |
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| Relations |
| BioSample |
SAMN13341806 |
| SRA |
SRX7196724 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4183386_BMI1_ChIP_PTC028_SF8628.tdf |
197.6 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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