|
| Status |
Public on Jun 02, 2016 |
| Title |
shCDK8, hypoxia, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
HCT116_shCDK8_hypoxia
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: colorectal carcinoma cell line
shRNA: CDK8
culture condition: 24 hrs hypoxia (1% O2)
|
| Treatment protocol |
untreated/normoxia: normal growth conditions; hypoxia: 24hrs 1% O2
|
| Growth protocol |
HCT116 cells were plated in McCoy's 5A medium supplemented with 10%FBS 24hrs prior to treatment
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using TRI reagent according to manufacturer's instructions and quality assessed using Bioanalyzer RNA Pico chips (Agilent). PolyA RNA was purified using the Dynabeads mRNA Direct micro kit (Life Technologies) according to manufacturer's instructions and ribosomal RNA contamination assessed using Bioanalyzer RNA Pico chips.
Libraries for Ion Torrent sequencing were prepared using the Ion Total RNAseq v2 kit (Life Technologies), according to manufactorer's instructions. Yeild and insert-size distribution were measured using Bioanalyzer High Sensitivity DNA chips (Agilent). The final Ion Torrent RNAseq libraries were subjected to an additional size-selection for 120-200bp on a Blue Pippin (Sage Science). Ion Torrent template preparation was carried out using the Ion PI Template OT2 200 Kit v2 and library sequencing carried out on an Ion Torrent Proton sequencer using the Ion PI Sequencing 200 Kit v2 (Life Technologies) according to manufacturer's instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
polyA RNA from HCT116 cells harboring CDK8 shRNA 24hrs hypoxic condition
|
| Data processing |
Signal processing, base calling and removal of barcode and adapter sequences was carried out automatically by the Torrent Suite Software (Life Technologies).
Data quality was assessed using custom scripts and the Fastx toolkit (version 0.0.13.2, hannonlab.cshl.fastx_toolkit.html). Reads shorter than 30 nt were removed and long reads trimmed to 150nt
Alignment to the human genome (hg19/GRCh37) was carried out using GSNAP (version 2013-10-28) with a mismatch setting of 3%.
Gene level counts were obtained using htseq-count (version 0.5.4p5) with a custom modified GTF annotation file based on an hg19 RefSeq gene list obtained from UCSC table browser (Karolchik et al., 2004).
Differential gene expression was assessed using DESeq (version 1.14.0) in R (version 3.0.3)
Genome_build: hg19/GRCh37
Supplementary_files_format_and_content: Gene level count data; tab-delimited text
|
| |
|
| Submission date |
Apr 27, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew D Galbraith |
| Organization name |
University of Colorado Anschutz Medical Campus
|
| Department |
Pharmacology & Linda Crnic Institute for Down Syndrome
|
| Street address |
RC1-N, Mail Stop 8303, Rm. P18-6114 12800 E. 19th Ave.
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL17303 |
| Series (1) |
| GSE68297 |
PolyA RNAseq from HCT116 cells in normoxia and hypoxia |
|
| Relations |
| BioSample |
SAMN03569172 |
| SRA |
SRX1011342 |
