|
| Status |
Public on Jan 02, 2013 |
| Title |
RNA_Seq_48hr_1 |
| Sample type |
SRA |
| |
|
| Source name |
RNA-seq H1-hESC-48hr-Activin
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: H1 embryonic stem cells
drug treatment: Activin
drug concentration: 50ng/ml
duration of treatment: 48hr
|
| Growth protocol |
H1 cells were grown to confluence in mTESR1 media. Cells were then differentiated for 48 hours in RPMI-B27 plus 50 ng/ml Activin
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was purified using mirVana miRNA isolation kit (Life Technologies) following the manufacturer instructions and treated with DNA-free™ DNase I (Ambion). 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 modified Illumina mRNA-Seq protocol. Polyadenylated RNA was fragmented and sequenced according to modified version of standard Illumina RNA-seq protocol. 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. RNA-seq libraries were sequenced on Illumina HiSeq 2000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNASeq_48hr_rep1
|
| Data processing |
For RNA-Seq samples. all sequenced reads were aligned to the human genome (Hg18) using the spliced read aligner TopHat version V1.4.0 (Trapnell et al. 2009). The paired end reads were aligned using "Bowtie -v" option for initial read mapping along with --microexon-search and --coverage-search options for identifying splicing.
supplementary_files_format_and_content: RPKM: The average (combining hESC or 48hr replicates) reads per kilobase of exonic length per million (RPKM) for each gene or lncRNA (see manuscript for details) was calculated and reported.
Images analysis and base calling was done using the solexa pipeline.
Genome_build: hg18
|
| |
|
| Submission date |
Sep 19, 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 |
GPL11154 |
| Series (1) |
| GSE41009 |
Divergent transcription of lncRNA/mRNA gene pairs in embryonic stem cells |
|
| Relations |
| Reanalyzed by |
GSE76586 |
| SRA |
SRX188847 |
| BioSample |
SAMN01179604 |
