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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 05, 2016 |
| Title |
RYBPfl/fl.YAF2KO_RING1B_UNT_rep1 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, Untreated control, RING1B ChIP
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| Organism |
Mus musculus |
| Characteristics |
cell type: RYBPfl/fl;YAF2-/- ES cells
treatment: none
chip antibody: RING1B (Cell Signaling, D22F2)
biological replicate: 1
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| Treatment protocol |
Cells were treated with 800nM 4-hydroxytamoxifen (OHT) for 96 hours prior to cell harvest.
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| Growth protocol |
RYBPfl/fl;YAF2-/- mouse embryonic stem cells were maintained in DMEM 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed for 1 hr in 2 mM EGS, followed by 15 min in 1% formaldehyde. Reactions were quenched by the addition of glycine to a final concentration of 125 µM. After cell lysis and chromatin extraction, chromatin was sonicated using a BioRuptor sonicator (Diagenode), followed by centrifugation at 16,000 x g for 20 min at 4°C, and the supernatant was taken for immunoprecipitation. Immunoprecipitations were performed overnight at 4°C using chromatin corresponding to 5 x 10^6 cells and were carried out in a total volume of 1 ml using approximately 3 µg of antibody. Antibody-bound chromatin was isolated on protein A agarose beads (RepliGen, Waltham, CA) or protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA). Beads were washed 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 in the presence of 200 mM NaCl where required. Samples were then treated with RNase and proteinase K before being purified with ChIP DNA Clean & Concentrator™ kit (Zymo, Irvine, CA).
Libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® kit, with 8-10 PCR cycles. Libraries were purified using AMPure XP cleanup, and quantified by qPCR using KAPA Library Quantitation Standards (KAPA Biosystems). Libraries were sequenced as 40bp paired-end reads on Illumina NextSeq 500 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Processed data file: mESC_RYBPfl.YAF2-KO_RING1B_UNT_DownsampledMERGED.MACS.bw
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| Data processing |
Paired-end reads were aligned to the mouse mm10 genome using bowtie2 (Langmead and Salzberg, 2012) with the "--no-mixed" and "--no-discordant" options, and non-uniquely mapping reads were discarded. PCR duplicates were removed using samtools and biological replicates were randomly downsampled to contain the same number of reads prior to being merged together.
Peaks were identified using MACS2 (--broad) for individual replicates, which were then combined. Only peaks identified in all biological triplicates were used.
Genome coverage tracks were made using the pileup function of MACS2 and visualised using the UCSC genome browser.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: BigWig files representing genome coverage for merged biological triplicates.
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| Submission date |
Jun 07, 2016 |
| 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 |
GPL19057 |
| Series (2) |
| GSE83094 |
RBYP stimulates PRC1 to shape chromatin-based communication between polycomb repressive complexes [ChIP-seq] |
| GSE83135 |
RBYP stimulates PRC1 to shape chromatin-based communication between polycomb repressive complexes |
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| Relations |
| BioSample |
SAMN05216867 |
| SRA |
SRX1831951 |
| 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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