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| Status |
Public on Oct 05, 2016 |
| Title |
RYBPfl/fl.YAF2KO_NativeInput_UNT_rep1 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells with Drosophila spike-in, Untreated control, MNase-digested chromatin control
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| Organism |
Mus musculus |
| Characteristics |
spiked-in organism: Drosophila melanogaster
cell type: RYBPfl/fl;YAF2-/- ES cells
treatment: none
chip antibody: none
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 |
A native ChIP protocol combined with a calibrated ChIP-seq approach was employed, as previously described (Hu et al., 2015; Bonhoure et al., 2014; Orlando et al., 2014). To achieve this, 1.25 x 10^7 Drosophila melanogaster S2 cells were spiked in to 5 x 10^7 Rybpfl/fl;Yaf2-/- mouse embryonic stem cells with or without OHT treatment. Nuclei were then isolated with RSB buffer (10 mM Tris-HCl (pH 8.), 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1% NP-40 and 5 mM N-ethylmaleimide, followed by MNase digestion for 5 min with 150 U MNase (Fermentas, Waltham, MA) in 1 ml RSB supplemented with 0.25 M sucrose, 3 mM CaCl2 and 10 mM N-ethylmaleimide. Digestions were stopped with EDTA, before nuclei were pelleted by centrifugation at 1500 x g and the soluble S1 fraction collected. Pelleted nuclei were then resuspended in 300 µl nucleosome release buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.2 mM EDTA, 10 mM N-ethylmaleimide), incubated at 4°C for 1 hr with gentle rotation, and then gently passed through a 27G syringe needle five times. Following centrifugation to pellet the insoluble material, the soluble S2 fraction was collected and combined with the S1 fraction. Immunoprecipitations were performed overnight at 4°C using chromatin corresponding to 5 x 10^6 cells. Antibody-bound chromatin was isolated on protein A agarose beads (RepliGen, Waltham, CA) or protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA). Native histone ChIPs were washed four times with native ChIP wash buffer (20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 125 mM NaCl and 0.1% Triton), followed by a final TE buffer wash. Samples were then treated with RNase and proteinase K before being purified with ChIP DNA Clean & Concentrator™ kit (Zymo, Irvine, CA). For calibrated ChIP-seq experiments, an input control was prepared for each individual sample, to allow the quantitation of spike-in consistency.
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 |
Calibrated ChIP-seq using spiked-in Drosophila melangaster S2 cells.
Processed data file: mESC_RYBPfl.YAF2-KO_NativeInput_UNT_mm10.NormalisedMERGED.MACS2.bw
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| Data processing |
ChIP-seq experiments which contained a spike-in genome (D. melanogaster) were aligned against a concatenated genome of the two genome sequences (mm10+dm6) using bowtie2 (Langmead and Salzberg, 2012) with the "--no-mixed" and "--no-discordant" options, and reads which mapped more than once were discarded. PCR duplicates were removed using samtools.
Spiking an identical concentration of D. melanogaster cells into our quantitative ChIP-seq experiments allows for calibration of each sample for IP efficiency and total mouse cell number. When comparing ChIP-seq for untreated and OHT-treated cell lines using an internal calibration, the number of mm10 reads were randomly downsampled to reflect the total number of dm6 reads in that sample. Furthermore, to adjust for variation in the dm6/mm10 ratio between biological replicates, each sample was adjusted using the percentage of dm6 reads relative to mm10 reads in each sample's input DNA.
Genome coverage tracks were made using the pileup function of MACS2 and visualised using the UCSC genome browser.
H2AK119ub1 peaks were identified using the dpeak function of DANPOS2 (-q 40 –kw 750 –kd 1500, height_logP > 100) (Chen et al., 2013) and peaks closer than 5 kb were merged.
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) |
| GSE83093 |
RBYP stimulates PRC1 to shape chromatin-based communication between polycomb repressive complexes [calChIP-seq] |
| GSE83135 |
RBYP stimulates PRC1 to shape chromatin-based communication between polycomb repressive complexes |
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| Relations |
| BioSample |
SAMN05216854 |
| SRA |
SRX1831939 |
| 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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