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| Status |
Public on May 23, 2023 |
| Title |
bioMBD2a-delGR_r1 |
| 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: Mbd3-KO
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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 |
Biotin ChIP: For cross-linking and chromatin extraction, cells were fixed for 10 minutes with 1% Formaldehyde at room temperature and incubated for 10 min on ice in presence of 1.2M Glycine. Cells were harvested and treated for 10 min with 10mM EDTA, 10mM TRIS, 0.5mM EGTA and 0.25% Triton X-100 and 10 min in 1mM EDTA, 10mM TRIS, 0.5mM EGTA and 200mM NaCl with subsequent lysis in 50mM HEPES, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.2% SDS and 300 mM NaCl for 2 hours on ice. Crosslinked chromatin was subjected to sonication in a Bioruptor Pico instrument (Diagenode) and diluted with equal volumes of 50mM HEPES, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate after successful sonication. ProteinA-dynabeads pre-cleared 150-250 µg chromatin were incubated with 40 µl blocked (1% CFSG, 100ng tRNA) Streptavidin-M280 magnetic beads over night at 4°C. Beads were washed with two rounds of 2% SDS, 1x high salt buffer, 1x LiCl Buffer and two rounds of TE. Beads were treated with RNaseA for 30 min at 37°C, Proteinase K for 3 hours at 50°C then de-crosslinked over night at 55°C. DNA was purified with Phenol-Chloroform extraction and EtOH precipitation.
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 using Bowtie directly executed from the R package QuasR with standard alignment parameters allowing up to 2 mismatches and excluding multi-mappers.
Coordinates of unique reads that map only once to the mouse genome were converted to WIG format for visualisation using qExportWig() with standard parameters in QuasR.
Assembly: mm9
Supplementary files format and content: wig track format: read coverage per consecutive 100bp windows
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| Submission date |
Feb 08, 2023 |
| Last update date |
May 24, 2023 |
| 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 (1) |
| GSE199541 |
Dissecting the roles of MBD2 isoforms and domains in regulating NuRD complex func-tion during cellular differentiation |
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| Relations |
| BioSample |
SAMN33214405 |
| SRA |
SRX19315891 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7034914_HA36CB1_mESC_MBD3KO_bioMBD2AGR_ChIP_46_1.wig.gz |
9.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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