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| Status |
Public on May 27, 2014 |
| Title |
KDM2Bfl/fl_RING1B_treated_rep3 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, tamoxifen treated, RING1B ChIP
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| Organism |
Mus musculus |
| Characteristics |
chip antibody: RING1B ab (Atsuta et al., 2001; PMID 11289226)
cell line: MS12 KDM2Bfl/fl ES cells
passage: p10-p20
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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 |
kdm2b fl/fl cell 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 and RNA-seq experiments, the cells were allowed to settle overnight without feeders and cultured in the absence or presence of 4-hydroxy tamoxifen (800 nM) for 72 hr to delete KDM2B ZF-CxxC domain.
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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 × 106 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 65oC, 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 triplicates for KDM2B and RING1B and biological duplicates for SUZ12. For mRNA-seq analysis 1 μg of purified total RNA was used to first isolate polyA plus RNA and then a directional library was prepared using the NEBNext UltraTM Directional RNA library Prep Kit for Illumina (NEB, Ipswich, MA).
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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| 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.
Paired end 51-bp reads from RNA-seq experiments were aligned to the mm9 reference genome using Tophat2 (v0.5) with default parameters.
FPKM values were obtained from biological triplicates of the Kdm2bfl/fl and 72 hr tamoxifen treated cells using cufflinks(v2.1.1) and cuffdiff(v2.1.1) on a refGene set obtained from the UCSC table browser.
genome build: mm9
processed data files format and content: Wig/BigWig files were created from merged BAM files using MACS. Tab delimited text files containing FPKM values for refGene gene IDs were obtained from Cufflinks.
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| Submission date |
Nov 21, 2013 |
| 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) |
| GSE52619 |
Deletion of KDM2B DNA-binding domain affects RING1B and SUZ12 occupancy |
| GSE55698 |
Variant PRC1 complex dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation |
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| Relations |
| BioSample |
SAMN02419017 |
| SRA |
SRX381489 |
| 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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