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| Status |
Public on Jan 25, 2022 |
| Title |
ChIP_H3K36me2_Setd2KO_rep1 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: TT2
cell type: Mouse embryonic stem cells
chip antibody: H3K36me2, Abcam ab9049
spike-in: Drosophila spike-in chromatin
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| Growth protocol |
Mouse embryonic stem cells and derivates were grown on gelatin coated dishes in DMEM containing 15% (v/v) fetal bovine serum, Penicillin streptomycin (10 000U/mL), 2mM L-Glutamine, 1X MEM Non-Essential Amino Acids, 10mM 2-Mercaptoethanol and 2mL of homemade Leukemia Inhibitory Factor (LIF) at 37°C with 5% C02. Setdb1 was knocked down by treating cells for 5 days with 800 nM 4-hydroxytamoxifen (4-OHT) in culture medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Two 15cm-dishes with 2.10^7 cells in 20mL of culture media were used for each ChIP-seq experiment. ChIP-seq was performed as described in ChIP-IT High sensitivity Kit from Active Motif with a few adaptations. Chromatin is sonicated by Bioruptor: 30sec ON – 30sec OFF, 10 cycles, high power. Sonicate for a total of 40 cycles for histones and of 30 cycles for TFs with 10min break on ice every 10 cycles. For quantitative ChIP reaction drosophila spike-in from Active Motif was used. For histone methylation of lysine 36, 25µg chromatin was used with 50ng Spike-in Chromatin + 4µg antibody + 2ug Spike-in Antibody. Spike-in antibody also required a blocking step. ChIP-DNA was eluted in 36µL with pre-warmed Elution Buffer then ChIP-DNA concentration was quantified with the Qubit dsDNA HS Assay Kit. 2,5ng to 10ng of ChIP-DNA were used for library preparation.
2,5ng to 10ng ChIP-DNA were used for library preparation. ChIP-seq library preparation was performed as described in TruSeq ChIP Sample Preparation kit from Illumina with few adaptations. The Enrich DNA Fragment step is proceeded before Purify Ligation Products step. AMPure XP beads were air-dry 7min. 1µL library was diluted 1:10 in H20 to quantified library concentration by Qubit DNA HS assay quantification. Agilent High Sensitivity DNA Electrophoresis was run to check DNA fragment size. Before sequencing, ChIP-seq libraries were quantified by qPCR. Libraries were sequenced 50bp or 100bp single-reads with 50 million reads of depth sequencing on HiSeq 2000 Illumina or NovaSeq 6000 Illumina machine at the MGX platforms.
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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 |
Quality controls of ChIP-seq Fastq files were performed by FastQC. ChIP-seq Fastq read files are mapped to the mm10 reference genome using Bowtie2 with default parameters. To determine for ChIP-seq peaks with a broad profile, datasets were processed using MACS2 with --broad and --g mm options. H3K36me3, H3K36me2 and H3K36me1 bigWig files were normalized using drosophila spike-ins, which were mapped to dm6 reference genome using Bowtie2 with default parameters. The normalization factor was determined as described in manufactured protocol (Spike-in Normalization from Active Motif). For each sample, normalized BigWig files were generated using Deeptools bamCoverage with --bs 50, --normalizeUsing RPKM and --scaleFactor options, where scaleFactor is the normalization factor specific to each sample.
Genome_build: mm10
Supplementary_files_format_and_content: bw files correpond RPKM-normalized bigwig binary files (50bp bins), which can be visualized using the Integrative Genomics Viewer (IGV) software.
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| Submission date |
Apr 08, 2021 |
| Last update date |
Jan 25, 2022 |
| Contact name |
Amandine BARRAL |
| Organization name |
IGH - CNRS
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| Lab |
Dejardin Lab
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| Street address |
141 rue de la cardonille
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| City |
Montpellier |
| State/province |
Hérault |
| ZIP/Postal code |
34090 |
| Country |
France |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE171749 |
SETDB1/NSD dependent H3K9me3/H3K36me3 dual heterochromatin maintains gene expression profiles by bookmarking poised enhancers |
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| Relations |
| BioSample |
SAMN18677752 |
| SRA |
SRX10559958 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5233350_ChIP_H3K36me2_Setd2KO_rep1.bw |
152.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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