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| Status |
Public on Jul 20, 2022 |
| Title |
H3K9me3_wt |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
genotype: wild type
antibody: H3K9me3
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| Growth protocol |
ES cells were were cultivated on feeder cells or 0.2% gelatine coated dishes. ES cell growth medium consisted of DMEM (Invitrogen) supplemented with 15% foetal calf serum (Invitrogen), 1x non-essential amino acids (Invitrogen), 1mM L-glutamine, LIF and 0.001% beta-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 15 x 10^6 cells were harvested per IP and fixed with 1 % methanol-free formaldehyde for 8 min. Cross-linking was quenched by adding glycine to a final concentration of 0.125 nM and incubated for 10 min at 4 ˚C on ice. Cells were pelleted at 600 x g for 5 min, washed with cold PBS and incubated for 10 min on ice in a buffer containing 10 mM EDTA, 10 mM Tris pH 8, 0.5 mM EGTA and 0.25 % Triton X-100. After centrifugation, cells were incubated in 1 mM EDTA, 10 mM Tris pH 8, 0.5 mM EGTA and 200 mM NaCl for 10 min on ice. Chromatin was extracted in a high salt buffer (HSB) containing 50 mM HEPES pH 7.5, 1 mM EDTA, 1 % Triton X-100, 0.1 % deoxycholate, 0.2 % SDS and 500 mM NaCl for 2 hours at 4 ˚C and chromatin was sheared using a Bioruptor Pico sonicator (Diagenode). 100 µg of chromatin was used per IP reaction with 30 µl of pre-blocked magnetic Protein A beads (Invitrogen). Beads were blocked with 1 mg of BSA and 100 ng of yeast tRNA (Sigma) in TE buffer containing proteinase inhibitor mix (Roche) before use. Prior to the IP, chromatin was pre-cleared with 20 µl of blocked beads for 1 hour at 4 ˚C. 5 % of input material was kept at -20 ˚C and de-crosslinked along the IP material. 5 µg of antibody was used per IP for overnight incubation at 4 ˚C. The next day, 30 µl of blocked beads were added to the chromatin and incubated for 4h at 4 ˚C. Beads were separated on a magnet and washed twice for 8 min with HSB, one time with 50 mM LiCl, 0.5 % NP-40, 0.5 % deoxycholate, 1 mM EDTA, and 10 mM Tris pH 8. After two additional washes with TE for 8 min, chromatin was eluted after 30 min incubation at 37 ˚C with 60 µg RNaseA (Roche) in 1 % SDS and 100 mM NaHCO3, followed by 3 h incubation adding 10 mM EDTA, 40 mM Tris pH 8 and 60 µg of Proteinase K (Roche). Final de-crosslinking was done overnight at 65 ˚C. Eluted material was cleaned up using phenol chloroform extraction and quantified using a Qubit 2.0 fluorometer (Thermo Scientific). The following antibodies were used for ChIP: H3K9me3 (Abcam ab8898 ).
ChIP-seq library using NEBNext ChIP-seq Library Prep Master Mix set for Illumina (NEB E6240)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ChIP
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| Data processing |
Reads were trimmed using trim_galore v0.6.6
Trimmed reads were aligned to the mm9 mouse genome BSgenome.Mmusculus.UCSC.mm9 using QuasR qAlign() in R with standard options
Wig files were created using QuasR qExportWig() function with scaling set to TRUE
Assembly: mm9
Supplementary files format and content: Wig track format: Read coverage per 100 bp bins
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| Submission date |
May 19, 2022 |
| Last update date |
Nov 29, 2022 |
| Contact name |
Tuncay Baubec |
| Organization name |
Utrecht University
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| Department |
Department of Biology
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| Lab |
Genome Biology and Epigenetics
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| Street address |
Padualaan 8
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| City |
Utrecht |
| ZIP/Postal code |
3584 CH |
| Country |
Netherlands |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE176459 |
DNA sequence determines epigenetic bistability at imprinting control regions [ChIP-Seq] |
| GSE176461 |
DNA sequence and chromatin modifiers determine epigenetic bistability at imprinting control regions |
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| Relations |
| BioSample |
SAMN28552670 |
| SRA |
SRX15378203 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6170906_MmES_K9me3_r2.wig.gz |
12.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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