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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 13, 2017 |
| Title |
ZHBTC4_ATAC_UNT_rep3 (ATAC-Seq) |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, untreated control, ATAC-seq
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| Organism |
Mus musculus |
| Characteristics |
cell line: ZHBTC4 (OCT4 conditional)
replicate: 3
passage: 30-40
treatment: untreated control
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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 accessibility was assayed using an adaptation of the assay for transposase accessible-chromatin (ATAC)-seq (Buenrostro et al., 2013). Briefly, 5 × 106 cells were harvested, washed with PBS and nuclei were isolated using 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05 % NP40 (IGEPAL CA630)), 1 mM PMSF, 3 mM DTT) for 1 min at room temperature. Nuclei were centrifuged at 1,000 × g for 5 min at 4°C, followed by a total of three washes with ice-cold RSB buffer (10mM NaCl, 10mM Tris (pH 7.4), 3mM MgCl2), to remove as much of contaminating cytoplasmic and mitochondrial material as possible. Nuclei were then counted, and 5×104 nuclei were resuspended in Tn5 reaction buffer (10mM TAPS, 5mM MgCl2, 10% dimethylformamide) and 2 µl of Tn5 transposase (25µM) made in house according to the previously described protocol (Picelli et al., 2014). Nuclei were then incubated for 30 min at 37°C, before isolation and purification of tagmented DNA using QiaQuick MinElute columns (Qiagen). To control for sequence bias of the Tn5 transposase, an ATAC “input” sample was generated, by tagmenting genomic DNA from ESCs with Tn5 for 30 min at 55°C.
ATAC-seq libraries were prepared by PCR amplification using custom made Illumina barcodes previously described (Buenrostro et al., 2013) and the NEBNext® High-Fidelity 2X PCR Master Mix with 8-10 cycles. Libraries were purified with two rounds of AMPure XP bead cleanup (1.5X beads:sample), followed by quantification by qPCR using SensiMix SYBR (Bioline) and KAPA Library Quantification DNA standards (KAPA Biosystems). ATAC-seq libraries were sequenced on Illumina NextSeq500 using 80bp paired-end reads in biological triplicate.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: ATAC-seq
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 Tn5 control provided as background.
Peakcalling was performed using the DANPOS2 dpeak function using untreated and treated samples in biological triplicate with Tn5 gDNA control digestion as background.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files representing genome coverage (with background subtracted) for merged biological triplicates were generated using DANPOS2.
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| Submission date |
Oct 11, 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) |
| GSE87819 |
The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells [ATAC-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 |
SAMN05894745 |
| SRA |
SRX2236890 |
| 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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