|
| Status |
Public on Jul 21, 2015 |
| Title |
SW480/pRTR-p53-VSV +DOX miRNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Colorectal cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
vector: pRTR-p53-VSV
treatment: 16 hours doxycyclin
cell line: SW480
|
| Treatment protocol |
SW480 cells were treated with DOX (100 ng/ml) for 16 hours to induce ectopic expression of p53.
|
| Growth protocol |
SW480 cells and their derivatives were cultured in DMEM grwoth medium containig 10% FBS at 37°C and 5%CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Trizol reagent (Life Technologies) following the manufacturer’s protocol. The small RNA fraction (representing lengths of about 18 to 25 nt) was gel-purified.
In short, an adenylated adapter was ligated to the 3’ end of the small RNA by a truncated T4 RNA Ligase 2, followed by the ligation of the 5’ adapter by T4 RNA Ligase 1 (NEB). After reverse-transcription with a specific primer, the cDNA was amplified by PCR. The correct PCR product was gel-purified, eluted in elution buffer, precipitated and solved in water. The quality of the libraries was assessed by qPCR and Bioanalyzer measurements.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiScanSQ |
| |
|
| Description |
small RNA sequencing data
|
| Data processing |
Libraries were sequenced on an Illumina HiScan (HiSeq Control Software v. 1.4; CASAVA 1.8)
Fastq files were processed with an in-house pipeline consisting of adapter trimming, read alignment, read counting and normalization. Adapters were trimmed by computing a suffix-prefix alignment of each read against the Illumina 3’ adapter sequence.
Trimmed reads were then aligned to the reference genome (hg19) using bowtie 0.12.7 allowing for up to 2 mismatches.
All best matches were retained and multi-mapping reads were equally divided among all mapping sites.
To compute miRNA read counts all reads mapping to annotated mature miRNA positions (according to mirbase v16) were considered. At their 3’ end, a tolerance of +/- 3 bp was allowed, but no tolerance at their 5’ end.
Normalization was performed by fitting a robust linear model to the quantile-quantile plot of log miRNA counts and taking the offset as normalization factor.
Genome_build: miRBase 16
Supplementary_files_format_and_content: miRNA read counts, txt-format
|
| |
|
| Submission date |
Mar 23, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Sabine Huenten |
| E-mail(s) |
sabine.huenten@med.uni-muenchen.de
|
| Organization name |
Ludwig-Maximilians-Universitaet Muenchen
|
| Street address |
Thalkirchner Strasse 36
|
| City |
Munich |
| ZIP/Postal code |
80336 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15456 |
| Series (2) |
| GSE67110 |
p53 in colorectal cancer cells |
| GSE67181 |
Genome-wide miRNA-expression analysis after p53 activation in colorectal cancer cells |
|
| Relations |
| BioSample |
SAMN03437981 |
| SRA |
SRX965481 |
