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| Status |
Public on May 27, 2014 |
| Title |
RING1A/B_cKO_H3K27me3_untreated_rep2 |
| Sample type |
SRA |
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| Source name |
RING1A-/-;RING1Bfl/fl Embryonic stem cells, untreated control, H3K27me3 ChIP
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| Organism |
Mus musculus |
| Characteristics |
cell type: Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 ES cells
passage: 15-25
chip antibody: H3K27me3 (Diagenode C15410069 (pAb-069)
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| Treatment protocol |
Chromatin was prepared as described previously (Farcas et al 2012), with minor modifications. Briefly, the cells were fixed for 1 hr in 2 mM EGS, followed by 15 min in 1% formaldehyde. Formaldehyde was quenched by the addition of glycine to a final concentration of 125 μM. Sonication was performed using a BioRuptor sonicator (Diagenode, Liege, Belgium) to produce fragments of approximately 0.5–1 kb. For RNA-Seq, total RNA was extracted using the Qiagen RNeasy Mini kit. Approximately 10 μg RNA was treated with Turbo DNase (Ambion, Carlsbad, CA) at 37°C for 30 min, according to the manufacturer's instructions. Genomic DNA-free RNA samples were further purified using the RNeasy kit RNA cleanup protocol. Samples were run on a 1% agarose gel to check quality of RNA preparation and integrity of 18S and 28S rRNA bands.
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| Growth protocol |
Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 ES cells were cultured on Mitomycin C inactivated mouse embryonic fibroblasts (MEFs) in DMEM (Gibco, Carlsbad, CA) supplemented with 15% FBS, 10 ng/mL leukemia-inhibiting factor (LIF), penicillin/streptomycin, beta-mercaptoethanol, l-glutamine and non-essential amino-acids on 0.1% gelatin-coated dishes. Prior to use in ChIPseq experiments, the cells were allowed to settle overnight without feeders and cultured in the absence or presence of 4-hydroxy tamoxifen (800 nM) for 48hr to delete RING1B.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To prepare material for ChIP-seq, immunoprecipitation was performed overnight at 4°C with approximately 3 µg of antibody and chromatin corresponding to 5 × 10^6 cells. Antibody bound chromatin was isolated on protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA) and washes were performed with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and TE buffer (x2) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To prepare ChIP-seq material, ChIP DNA was eluted, and cross-links reversed at 65°C, then samples were then sequentially treated with RNase and proteinase K before being purified on a PureLink PCR micro column (Invitrogen). ChIP sequencing libraries were generated as described previously (Blackledge et al., 2010) and sequenced on the Illumina HiSeq2000 platform with 51 bp reads. ChIP sequencing experiments were performed in biological duplicates with matched input controls.
Performed by the High-Throughput Sequencing centre at the Wellcome Trust Centre for Human Genetics (Oxford, United Kingdom) according to standard Illumina library generation protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RING1AB_cKO_H3K27me3_untreated_merged.bigwig
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| Data processing |
Single-end 51bp reads from ChIP-seq experiments were mapped to mm9 using bowtie v1.1.2. Reads which could be aligned to more than one site in the genome were supressed (-m 1).
Duplicate reads were removed. Biological replicates were down-sampled using Picard (v1.97) to the same total read count (of mapped reads).
MACS(v1.4) was used to call peaks for each biological replicate with the control input sample. Peaks from different biological replicates were merged using Bedops (v2.2.0) and intervals not represented in all independent replicates were eliminated.
Genome_build: mm9
Supplementary_files_format_and_content: Wig/BigWig files were created from merged BAM files.
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| Submission date |
Mar 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Hamish W King |
| Organization name |
Queen Mary University of London
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| Department |
Blizard Institute
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| Street address |
4 Newark St
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| City |
London |
| ZIP/Postal code |
E1 2AT |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE55697 |
Deletion of RING1A/B and loss of H2AK119ub1 affects PRC2 occupancy and H3K27me3 genome-wide |
| GSE55698 |
Variant PRC1 complex dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation |
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| Relations |
| BioSample |
SAMN02677587 |
| SRA |
SRX482889 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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