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| Status |
Public on Jul 08, 2018 |
| Title |
G34R_H3K36me3_ChIPSeq |
| Sample type |
SRA |
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| Source name |
ES Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Targeted H3.3 G34R ES cells
passages: < 20
strain: 129S1/SvImJ
chip antibody: H3K36me3 antibody (Abcam)
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| Growth protocol |
Mouse ESCs were cultured in Dulbecco's modified Eagle's medium supplemented with 15% heat-inactivated foetal calf serum, 10e3 units/ml leukemia inhibitory factor and 0.1 mM β-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 million cells were fixed in growth media with 0.4% formaldehyde for 10 min and quenched with 125 mM glycine. Chromatin was released by sequential lysis with cells lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% Igepal) and nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS). Chromatin was sheared to ≈300 bp with 12 rounds of sonication (30 s on, 30 s off) on a Bioruptor (Diagenode) and incubated overnight at 4 ᵒC with either 5 μg H3K9me3 antibody (Abcam ab8898) or 10 μg H3K36me3 antibody (Abcam??). Samples were immunoprecipitated with Protein A Agarose beads (Sigma-Aldrich, 05015979001) and washed sequentially with low salt (20 mM Tris pH 8, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), high salt (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), and LiCl buffers (10 mM Tris pH 8, 1 mM EDTA, 0.25 M LiCl, 1% Igepal, 1% sodium deoxycholate). Chromatin was eluted with elution buffer (1% SDS, 100 mM NaHCO3), decrosslinked overnight at 65ᵒC, incubated with Proteinase K, phenol chloroform extracted and ethanol precipitated.
Sample concentrations were determined by Qubit (ThermoFisher Scientific) and 20 ng of DNA was used as starting material. ChIP libraries were prepared with Nugen Ovation Ultralow System V2 (Nugen protocol M01379v1, 2014) with 10 cycles of amplification. Libraries quality was assessed by Qubit, Bioanalyzer (Agilent) and qPCR, and a single equimolar pool was made based on size adjusted qPCR quantitation. Following denaturation, 12 pM of library pools were used for cBot hybridisation and cluster generation (Illumina Protocol 15006165 v02 Feb 2016), and samples were sequenced on an Illumina HiSeq 1500 rapid mode (50 bp SR sequencing, Illumina Protocol 15035788 Rev D, Apr 2014).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
ChIP-seq of H3K36me3 and H3K9me3 from WT and H3.3 G34R mutants were aligned to the mouse genome (mm9) with Bowtie (v1.0.0) with default settings except (m -5) to allow restricted multi-mapping to short repeats.
Data were imported into Seqmonk (v 0.32.1) with PCR duplicates excluded. Reads were counted across 200 bp genomic bins and samples were normalised for total reads (per million reads). WT and H3.3 G34R datasets were normalised against each other using the “match distribution quantitation” tool on Seqmonk.
Selected bins which overlapped UCSC annotated genes and only analysed sites which were enriched for H3K36me3/H3K9me3 (>95th percentile) in either WT or G34R cells. All windows within 100 bp were merged using MergeBed (-d 100 -o mean) function in BedTools (v 2.20.1). Merged regions >500 bp and regions with extreme read counts (top 0.01%), typically associated with poorly annotated genomic regions, were excluded from further analysis. A total of 31,593 WT/G34R H3K36me3 enriched sites and 32,502 WT/G34R H3K9me3 enriched sites were used for downstream analyses.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files were generated with Wig/BedGraph-to-bigWig (v1.1.0) in Galaxy with default parameters. Scores represent normalised read counts in 200 bp bins.
Supplementary_files_format_and_content: Tab delimited text files represent regions which were deemed to be enriched for either H3K36me3 or H3K9me3.
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| Submission date |
Oct 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Hsiao Phin Joanna Voon |
| E-mail(s) |
Joanna.Voon@monash.edu
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| Organization name |
Monash University
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| Department |
Biochemistry and Molecular Biology
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| Lab |
Epigenetics and Chromatin
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| Street address |
Monash University, Wellington Road
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| City |
Clayton |
| State/province |
Victoria |
| ZIP/Postal code |
3800 |
| Country |
Australia |
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| Platform ID |
GPL18480 |
| Series (2) |
| GSE106203 |
H3K9me3 and H3K36me3 ChIP-seq in WT and H3.3 G34R mouse ES cells |
| GSE106205 |
The H3.3 G34R glioma-associated mutation alters H3K9me3 and H3K36me3 chromatin modifications |
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| Relations |
| BioSample |
SAMN07837509 |
| SRA |
SRX3329727 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2831542_G34R_H3K36me3_aligned.bw |
80.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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