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| Status |
Public on Mar 28, 2022 |
| Title |
H3K27me3_ChIP-seq_mESC_Parental |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells & S2 cells
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
chip antibody: H3K27me3 (Cell Signaling Technologies #9733)
cell line: ESC line V6.5 1A
cell type: C57BL/6 x 129S4/SvJae F1 embryo-derived embryonic stem cells
genotype: wild-type
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| Growth protocol |
Mouse embryonic stem cells were maintained on gelatin-coated plates in Knockout DMEM (Gibco) supplemented with 15% ES-cell-qualified FBS (Gemini), 0.1 mM 2-mercapoethanol, 2mM L-glutamine (Life technologies) and LIF. Drosophila S2 cells were cultured in Schneider’s Drosophila Medium (Invitrogen) containing 10% heat-inactivated FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To obtain a soluble chromatin extract, ~2x10^7 cells were resuspended in 1 mL LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete protease inhibitor) and incubated rotating at 4°C for 10 min. Samples were centrifuged, resuspended in 1 mL LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Compete protease inhibitor), and incubated rotating at 4°C for 10 min. Finally, samples were centrifuged, resuspended in 1 mL LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100, 1x Complete protease inhibitor), and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 8 min (anti-HA ChIP) or 12 min (anti-histone PTM ChIP) using a Covaris E220 focused ultra-sonicator at peak power 140, duty factor 5, and cycles/burst 200. For histone PTM ChIP-Rx, after centrifugation soluble chromatin was spiked-in with soluble chromatin from Drosophila S2 cells that was similarly prepared and equivalent to 5-10% of the mouse/human cell chromatin. The lysates were incubated with 100 μl Pierce anti-HA beads (Themo Scientific, 88836) or with anti-H3K4me1 (Abcam, ab8895), anti-H3K9me3 (Abcam, ab8898), anti-H3K27ac (Active Motif, 39133), anti-H3K27me3 (Cell Signaling Tech, 9733), anti-H3K36me2 (Cell Signaling, 2901) or anti-H3K36me3 (Active Motif, 61101) antibody bound to 75 μl protein A or protein G Dnya1 magnetic beads (Invitrogen) and incubated overnight at 4°C with 5% kept as input DNA. Magnetic beads were washed with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), LiCl buffer (150 mM LiCl; 0.5% Na deoxycholate; 0.1% SDS; 1% Nonidet P-40; 1 mM EDTA; 50 mM Tris-HCl) and TE buffer (1 mM EDTA; 10 mM Tris-HCl). For ChIP-seq, beads were resuspended in elution buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10mM EDTA, 200 mM NaCl) and incubated for 30 min at 65°C. After centrifugation the eluate was reverse cross-linked overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C and with Proteinase K (Roche) for 1 hr at 55°C and DNA was recovered using Qiagen PCR purification kit.
KAPA Hyper Prep Kit
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
According to manufacturer's protocol
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| Data processing |
Raw ChIP sequencing reads were aligned using BWA version 0.7.17 with default parameters
Raw RNA sequencing reads were aligned using STAR version 2.5.3a with default parameters
Whole-Genome Bisulfite Sequencing (WGBS) raw reads were submitted to Bismark(version 0.22.3) for mapping and methylation calling, against mouse genome version mm10/GRCm38, discarding duplicate reads. Briefly, the methylation level at a CpG (similarly for CHH) site was the number of reads with that site methylated divided by the total number of reads covering the site. To ensure comparability of region DNA methylation levels across all samples, CpGs that overlapped with SNPs from dbSNPs or were located within the ENCODE blacklisted regions, termed DER (Duke excluded regions) were excluded and only CpGs covered by ≥ 5x in all samples were retained for the computation of DNA methylation levels. We defined the average methylation for a genomic region as the coverage-weighted mean of the methylation levels of the individual CpGs within the region.
Genome_build: mm10 and dm6 for mouse and drosophila data, respectively
Supplementary_files_format_and_content: bigWig, tdf
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| Submission date |
Oct 25, 2021 |
| Last update date |
Mar 30, 2022 |
| Contact name |
Jacek Majewski |
| E-mail(s) |
jacek.majewski@mcgill.ca
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| Organization name |
McGill University
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| Department |
Human Genetics
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| Street address |
740 Dr. Penfield
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
QC H3A 1A4 |
| Country |
Canada |
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| Platform ID |
GPL25475 |
| Series (1) |
| GSE186506 |
H3K36 Dimethylation Shapes the Epigenetic Interaction Landscape by Directing Repressive Chromatin Modifications in Embryonic Stem Cells |
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| Relations |
| BioSample |
SAMN22560408 |
| SRA |
SRX12764386 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5653814_mESC_Par_H3K27me3.bw |
91.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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