|
| Status |
Public on Jan 02, 2013 |
| Title |
RNA-Seq 48HR |
| Sample type |
SRA |
| |
|
| Source name |
ZHBTc4 ES cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6-129
cell type: Embryonic stem cells
concentration: 2 ug/mL
drug: dox
duration of treatment: 48 hr
|
| Growth protocol |
ZHBTc4 Oct4 shutdown inducible cells were grown on irradiated MEFs and split onto gelatin coated dishes for 2 passages in mES media. Oct4 shutdown was induced by addition of 2ug doxycycline for 48 hours.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was purified using mirVana miRNA isolation kit (Life technologies) following the manufacturer instructions. Synthetic RNAs (ERCC ExFold RNA Spike-In kit, Ambion, 4456739) were added to each population based on cell number. See manuscript for details. Polyadenylated RNA was purified from the total RNA by two rounds of selection using Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA (Life Technologies) following the manufacturer instructions and sequenced using a standard Illumina mRNA-Seq protocol with the following modifications. Briefly, polyadenylated RNA was fragmented with divalent cations under elevated temperature. First strand cDNA synthesis was performed with random hexamers and Superscript III reverse transcriptase. Second strand cDNA synthesis was performed using RNAse H and DNA Polymerase I. In the second-strand synthesis reaction, dTTP was replaced with dUTP. Following cDNA synthesis, the double stranded products were end repaired, a single “A” base was added, and Illumina PE adaptors were ligated onto the cDNA products. The ligation products with an average size of 300 bp were purified using agarose gel electrophoresis. Following gel purification, the strand of cDNA containing dUTP was selectively destroyed during incubation of purified double-stranded DNA with HK-UNG (Epicentre). The adapter ligated single-stranded cDNA was then amplified with 15 cycles of PCR and PCR products were purified using gel electrophoresis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA-Seq from ZHBTc4 cells after 48HR of treatment
Sample name: L22_1538
|
| Data processing |
Images analysis and base calling was done using the solexa pipeline.
For all ChIP-Seq samples reads were aligned to their indicated build using bowtie with parameters -e 70 -k 1 -m 1 -n 2 --best --sam. Seed length (-l) was set to read length for each dataset.
For all RNA-Seq samples reads were aligned to their indicated build using bowtie with parameters -e 70 -k 1 -m 2 -n 2 --best --sam. Seed length (-l) was set to read length for each dataset.
Genome_build: mm9
Supplementary_files_format_and_content: WIG files: For all ChIP-Seq samples, wiggle files were produced by MACS 1.4 with --space=50.
Supplementary_files_format_and_content: RNA-Seq RPKM expression values were computed for each gene and normalized to the included ERCC controls. Details can be found with the associated manuscript.
|
| |
|
| Submission date |
Nov 26, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard A Young |
| E-mail(s) |
young_computation@wi.mit.edu
|
| Phone |
617-258-5219
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Young Lab
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE42474 |
Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes [ChIP-Seq and RNA-seq] |
| GSE44288 |
Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes |
|
| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX206373 |
| BioSample |
SAMN01818828 |
