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| Status |
Public on May 13, 2019 |
| Title |
ML_HGG_005_Xn_input |
| Sample type |
SRA |
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| Source name |
H3.3K27WT_Xenograft
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| Organism |
Homo sapiens |
| Characteristics |
genotype: H3.3K27WT
sample type: Xenograft
chip antibody: None
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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 4000 |
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| 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
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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 |
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] |
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| Relations |
| BioSample |
SAMN11309318 |
| SRA |
SRX5620660 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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