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| Status |
Public on Mar 28, 2022 |
| Title |
H3K27me1_ChIP-seq_mESC_DNMT_TKO |
| 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: H3K27me1 (Active Motif #61015)
cell line: ESC line J1
cell type: 129S4/SvJa embryo-derived embryonic stem cells
genotype: Dnmt1-/-;Dnmt3a-/-;Dnmt3b-/-
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| Growth protocol |
Cells were cultured in Knockout-DMEM (Gibco) with 15% ES qualified FBS (Gibco), ESGRO mouse LIF 1000U/mL (Millipore), 1X PenStrep (Invitrogen), 1X Glutamax (Invitrogen), 55µM b-merceptoethanol (Invitrogen), 1X non-essential amino acids (Invitrogen) and 1X Primocin (Invivogen). Cells were maintained at 37 °C and 5% CO2 on a layer of murine embryonic feeders (MEFs). Prior to collection for ChIP experiments, cells were passaged once on plates pretreated with 0.1% gelatin but without feeders. The day of collection, cells were washed with PBS followed by digestion with TrypLE 30% (for A1 lines) or trypsin 0.25% (for J1 lines) for 7 minutes at 37 °C. Trypsinizaton was terminated by addition of 1X SoyBean Trypsin inhibitor (for A1 lines) or of media (for J1 lines).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 10 million cells per cell line were grown and directly crosslinked on the plate with 1% formaldehyde (Sigma) for 10 minutes at room temperature and the reaction was stopped using 125nM Glycine for 5 minutes. Fixed cell preparations were washed with ice-cold PBS, scraped off the plate, pelleted, washed twice again in ice-cold PBS, and flash frozen pellets stored at −80°C. Thawed pellets were resuspended in 500ul cell lysis buffer (5 mM PIPES-pH 8.5, 85 mM KCl, 1% (v/v) IGEPAL CA-630, 50 mM NaF, 1 mM PMSF, 1 mM Phenylarsine Oxide, 5 mM Sodium Orthovanadate, EDTA-free Protease Inhibitor tablet) and incubated 30 minutes on ice. Samples were centrifugated and pellets resuspended in 500ul of nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS, 50 mM NaF, 1 mM PMSF, 1 mM Phenylarsine Oxide, 5 mM Sodium Orthovanadate and EDTA-free protease inhibitor tablet) and incubated 30 minutes on ice. Sonication of lysed nuclei was performed on a BioRuptor UCD-300 at max intensity for 60 cycles, 10 s on 20 s 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 of a reversed cross-linked and purified aliquot. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and concentrated using Nanosep 10k OMEGA (Pall). Before ChIP reaction 2% of sonicated Drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing. ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit for Histones. Dynabeads Protein A (Invitrogen) were washed, then incubated with specific antibodies, 1.5 million cells of sonicated cell lysate, and protease inhibitors for 10 hr, followed by 20 min wash cycle using the provided wash buffers (Diagenode Immunoprecipitation Buffers, iDeal ChIP-seq kit for Histone). Reverse cross-linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail at 65°C for 30 min followed by 2ul Proteinase K at 65°C for 30 min. Samples were then purified with QIAGEN MinElute PCR purification kit (QIAGEN) as per manufacturers’ protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse cross-linked and DNA was isolated following the same protocol.
KAPA Hyper Prep Kit
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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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 |
GPL29685 |
| 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 |
SAMN22560437 |
| SRA |
SRX12764410 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5653835_mESC_DNMT_TKO_H3K27me1.bw |
487.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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