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Sample GSM3701084 Query DataSets for GSM3701084
Status Public on May 13, 2019
Title DIPGXIII-ko-c10p5-Rx_R2_cells_h3k27ac
Sample type SRA
 
Source name H3.3K27M-KO_Tumor-derived cell-line
Organism Homo sapiens
Characteristics genotype: H3.3K27M-KO
sample type: Tumor-derived cell-line
chip antibody: Diagenode-H3K27ac (Cat# C15410196, RRID:AB_2637079)
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
 
Data processing Sequence reads were aligned to the human genome (HG19) using Bowtie2 (v2.1.0) (Langmead and Salzberg, 2012) under default settings. PCR duplicates were removed using PICARD tools generating .BAM files (Li et al., 2009). Read processing and alignment for analysis of repeat elements was performed separately (see below for details). Significant peaks were identified using Model-Based Analysis for ChIP-seq (MACS 1.4) (Zhang et al., 2008)with a p value cutoff of 1e-9. Peaks were annotated using HOMER (v3.12) (Heinz et al., 2010) with promoter regions classified as any peak within +/- 2.5 kb of a transcriptional start site (TSS), and enhancer region greater than 2.5 kb from a TSS. Peaks were also annotated using ChIP-atlas annotating distal enhancers to genes based upon public CHIA-PET datasets. Super enhancers were identified using the ROSE algorithm with exclusion of peaks within +/- 2.5 kb of a TSS and a stitch distance of 12.5 kb. For visualization of H3K27 acetylation profiles, BAM alignment files were normalized to RPKM values using DeepTools (Ramirez et al., 2014) and visualized in Integrated Genome Viewer (v2.3.40).
Genome_build: hg19
 
Submission date Apr 01, 2019
Last update date May 13, 2019
Contact name Nada Jabado
Organization name McGill University
Department Department of Pediatrics
Lab Jabado Lab
Street address 1001 Décarie Boulevard
City Montreal
State/province Québec
ZIP/Postal code H4A 3J1
Country Canada
 
Platform ID GPL20301
Series (2)
GSE128745 Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas
GSE129135 Pervasive H3K27 acetylation leads to ERV expression and a therapeutic vulnerability in H3K27M gliomas [ChIP-Seq]
Relations
BioSample SAMN11309299
SRA SRX5620630

Supplementary file Size Download File type/resource
GSM3701084_DIPGXIII-ko-c10p5-Rx_cells_h3k27ac_merge_realigned_peaks.bed.gz 333.3 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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