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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 13, 2017 |
| Title |
ZHBTC4_nucRNA_UNT_rep1 (RNA-Seq) |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, untreated control, nucRNA-seq
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| Organism |
Mus musculus |
| Characteristics |
cell line: ZHBTC4 (OCT4 conditional)
replicate: 1
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 |
total RNA |
| Extraction protocol |
1 × 10e7 cells were resuspended in 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 to isolate nuclei. 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. Nuclei were then resuspended in 1mL of TriZOL reagent (ThermoScientific) according to the manufacturer’s protocol. Nuclear RNA was treated with the TURBO DNA-free Kit (ThermoScientific) and depleted for rRNA using the NEBNext rRNA Depletion Kit (NEB).
RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB) and libraries were sequenced on the Illumina NextSeq500 with 80bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
RNA-seq reads were initially aligned against the rRNA genomic sequence (GenBank: BK000964.3) using bowtie2 to filter out rRNA fragments, prior to alignment using the STAR RNA-seq aligner (Dobin et al., 2012). To improve mapping of nascent, intronic sequences, reads which failed to map using STAR were aligned against the genome using bowtie2.
Biological triplicate read counts were determined using custom scripts for a custom-built, non-redundant mm10 gene set. Briefly, mm10 refGene genes were filtered on size (>200bp), gene body and TSS mappability, unique TSS and TTS, so as to remove poorly mappable and highly similar transcripts.
The final set of 20,633 genes were used for differential analysis using DESeq2 (Love et al., 2014), with default settings and a significance threshold of padj < 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: [.bw] Stranded bigWig files representing genome coverage for merged biological triplicate
Supplementary_files_format_and_content: [.txt] ReadCounts and DESeq2 results
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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) |
| GSE87821 |
The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells [RNA-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 |
SAMN05894797 |
| SRA |
SRX2236945 |
| 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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