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| Status |
Public on May 13, 2019 |
| Title |
DIPGXIII-C14_H3K27ac |
| Sample type |
SRA |
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| Source name |
H3.3K27M_Tumor-derived cell-line
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| Organism |
Homo sapiens |
| Characteristics |
genotype: H3.3K27M
sample type: Tumor-derived cell-line
chip antibody: H3K27ac antibody (Diagenode, C15410196)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at -80°C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 60 cycles, 10s on 20s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and before ChIP reaction ~5% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing (see below). ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. 25ul Protein A beads were washed and then incubated with 6ug of H3K27ac antibody (Diagenode, C15410196), and 2 million cells of sonicated cell lysate combined with protease inhibitors for 10 hr, followed by 20 min wash cycle with provided wash buffers. Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail (Life Technologies) at 65°C for 30 min followed by 2ul Proteinase K (Thermo Fisher Scientific) at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers’ protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol.
Library preparation was carried out using Kapa HTP Illumina library preparation reagents. Briefly, 25ul of ChIP sample was incubated with 45ul end repair mix at 20°C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50ul buffer enzyme mix for 30°C 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead bound sample was incubated with 45ul buffer enzyme mix and 5ul of different TruSeq DNA adapters (Illumina) for each sample, for 20°C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6x/0.8x ratio of Ampure XP beads (double size selection) set to collect 250-450bp fragments.
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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 |
Adaptor sequences and the first four nucleotides of each read were removed from the read sets using Trimmomatic (Bolger et al., 2014) (v0.32). Reads were scanned from start to end and truncated if and when the average quality of a 4-nucleotide sliding window fell too low (phred33<30). Short reads (<30 bp) were subsequently discarded. Multiple quality control metrics were obtained using FASTQC (v0.11.2), samtools (Li et al., 2009) (v0.1.19), BEDtools (Quinlan and Hall, 2010) (v2.17.0) and custom scripts. Reads were processed as described for RNAseq, and then aligned using the human repeat genome as reference, as previously described (Solovyov et al., Cell Reports 2018). Repbase reference genome was used (Bao et al., 2015) (v23.03) (http://www.girinst.org/repbase). Since Repbase largely consists of family consensus sequences, reads are assigned to these sequences regardless of where and how many times they are present in the genome, mitigating mapping uncertainty. We combined humrep.ref and humsub.ref into a single reference of repeat sequences for the human genome, covering a total of 1132 consensus elements, and counted the number of reads mapping into each of these elements using featureCounts (MAPQ>3). Differential enrichment of H3K27ac at the consensus sequences were then calculated using DESeq2 (Love et al., 2014). The inverse of ChIP-Rx spike-in scaling factors (i.e. 1/rx) were supplied to DESeq2 as normalization factors (i.e. size factors).
Genome_build: hg19
Supplementary_files_format_and_content: tab separated values containing raw read counts
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| Submission date |
Apr 01, 2019 |
| Last update date |
May 13, 2019 |
| Contact name |
Nada Jabado |
| Organization name |
McGill University
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| Department |
Department of Pediatrics
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| Lab |
Jabado Lab
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| Street address |
1001 Décarie Boulevard
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| City |
Montreal |
| State/province |
Québec |
| ZIP/Postal code |
H4A 3J1 |
| Country |
Canada |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE128745 |
Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas |
| GSE129136 |
Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas [ChIP-Seq 2] |
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| Relations |
| BioSample |
SAMN11309349 |
| SRA |
SRX5620677 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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