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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 13, 2017 |
| Title |
BRG1fl_BRG1_72hTAM_rep3 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, untreated control, BRG1 ChIP-seq
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| Organism |
Mus musculus |
| Characteristics |
cell line: Brg1fl/fl (BRG1 conditional)
ChIP: BRG1
antibody: Abcam (ab110641)
replicate: 3
passage: 15-25
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| Treatment protocol |
ZHBTC4 cells were treated with 1 µg/mL doxycycline for 24 h to ablate OCT4 expression. Brg1fl/fl ESCs were treated with 4-hydroxytamoxifen for 72 h to ablate BRG1 protein levels.
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| Growth protocol |
Mouse embryonic stem cells containing a doxycycline-sensitive OCT4 transgene (ZHBTC4; (Niwa et al., 2000)) were grown on gelatin-coated plates in DMEM (Gibco, Carlsbad, CA) supplemented with 15 % FBS, 10 ng/mL leukemia-inhibitory factor, penicillin/streptomycin, beta-mercaptoethanol, l-glutamine and non-essential amino-acids. Brg1fl/fl ESCs were previously described (Ho et al., 2011) and were maintained in DMEM KnockOut supplemented with 10 % FBS and 5 % serum replacement, plus additional factors described for ZHBTC4 ESCs.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed as described previously (Farcas et al., 2012), with minor modifications. Cells were fixed for 1 h in 2 mM DSG and 12.5 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 × g for 20 min at 4°C, and used fresh or stored at -80°C. Chromatin was quantified by denaturing chromatin 1:10 0.1 M NaOH, and measuring DNA concentration by NanoDrop and 150 µg chromatin/IP was diluted ten-fold in ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl (pH 8), 150 mM NaCl) prior to pre-clearing with prepared protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA) which had been blocked for 1 h with 0.2 mg/mL BSA and 50 µg/mL yeast tRNA. Antibody-bound chromatin was isolated on protein A magnetic Dynabeads. ChIP 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 % sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and TE buffer (x2 washes) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA).
To prepare ChIP-seq material, ChIP DNA was eluted using 1 % SDS 100 mM NaHCO3, and cross-links reversed at 65°C in the presence of 200 mM NaCl. Samples were then treated with RNase and proteinase K before being purified with ChIP DNA Clean & Concentrator™ kit (Zymo, Irvine, CA). ChIP-seq libraries were prepared using the NEBNext Ultra DNA Library Prep Kit, and sequenced as 38bp paired-end reads on Illumina NextSeq500 platform. All ChIP-seq experiments were carried out in biological triplicate.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
For ATAC-seq and ChIP-seq, paired-end reads were aligned to the mouse mm10 genome using bowtie2 (Langmead and Salzberg, 2012) with the “—no-mixed” and “—no-discordant” options. Non-uniquely mapping reads and reads mapping to custom “blacklist” of artificially high regions of the genome were discarded. PCR duplicates were removed using SAMtools (Li et al., 2009). Biological replicates were randomly downsampled to contain the same number of reads for each individual replicate and merged.
Merged BAM files were used to make a genome track using DANPOS2, with the input control provided as background.
Peakcalling was performed using the DANPOS2 dpeak function using untreated and treated samples in biological triplicate with input control as background.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files representing genome coverage (with background subtracted) for merged biological triplicate were generated using DANPOS2.
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| Submission date |
Mar 03, 2017 |
| 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) |
| GSE87820 |
The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells [ChIP-Seq] |
| GSE87822 |
The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN06475102 |
| SRA |
SRX2612048 |
| 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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