|
| Status |
Public on Feb 13, 2014 |
| Title |
MCF7_Normoxia_microRNAseq_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF-7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 21 percent Oxygen
time: 48h
cell line: MCF7
|
| Treatment protocol |
Exposure of cell cultures to hypoxia (1% oxygen) was undertaken in an In vivo2 Hypoxia Work Station (Ruskin Technologies) in parallel with cells maintained in normoxic conditions (21% oxygen). Cells were recovered after 16h, 32h and 48h of exposure to hypoxia.
|
| Growth protocol |
MCF-7 cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 2mmol/L of L-glutamine.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cells using the miRVana miRNA isolation kit (Ambion) in accordance with the prescribed protocol provided with the kit. RNA quality was assessed after extraction using Agilent bioanalyser.
Libraries were prepared from the 20 to 30 nucleotide RNA fraction according to the Illumina DGE-small RNA sample preparation protocol (v1.5). The 20 to 30 nucleotide RNA fraction was isolated from 2µg of total RNA after being run in a 15% urea-TBE gel (Invitrogen) for 1h at 200V. The RNA contained on the excised band was eluted in 300µl of 0.3M NaCl by 4h of constant rotation at room temperature. The elute was separated from the gel debris using a Spin-X-column (Thermo Fisher Scientific) and RNA was precipitated by adding 750µl of 100% ethanol and 3µl of glycogen (1mg/ml) and incubating for 30 minutes at -80˚C. The precipitated RNA was centrifuged at 14,000 rpm for 25 minutes at 4˚C, washed with 75% ethanol, air dried and resuspended in 5µl of RNAse-free water. This material was used to prepare miRNA sequencing libraries using the DGE-small RNA sample preparation kit (v1.5) from Illumina and according to the protocol provided with the kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Total RNA containing short RNA fraction
|
| Data processing |
Small RNA sequencing data were processed from raw FASTQ files and analysed using the Kraken pipeline (http://www.ebi.ac.uk/research/enright/software/kraken)
The 3' adaptors were stripped using Kraken tools and according the following criteria: 12-nt alignment stretch with no more than 2 mismatches and no gaps.
The reads were filtered for low-complexity regions and the remaining reads were size-selected for 18-26nt, all using Kraken tools. A file with unique reads and their corresponding counts was generated for each sample.
The final processed unique reads were mapped to the human genome (hg19 based on Ensembl v64) using bowtie 0.12.7 allowing for two mismatches and using the best alignment stratum option. Reads mapped to more than 20 loci were discarded. Mapped reads were classified and counted based on genomic annotations (RNAs, coding genes, pseudogenes and repeats) obtained from Ensembl v64. For reads mapping to multiple loci, counts were divided by total number of reads.
For differential expression of miRNAs, the overlap between mapped reads and human mature miRNAs (based on miRBase v17) was found using the function findOverlaps available in the Bioconductor GenomicRanges package (http://www.bioconductor.org/packages/2.10/bioc/html/GenomicRanges.html). The normalization and differential expression was performed with Bioconductor edgeR package v2.3.57 using both common and tagwise dispersion. The significant differentially expressed miRNAs were determined by an adjusted P value <0.05 based on the Benjamini and Hochberg multiple testing correction.
Genome_build: hg19 based on Ensembl v64 and miRBase (human, v17)
Supplementary_files_format_and_content: Normalised counts using the TMM method of edgeR package from Bioconductor
|
| |
|
| Submission date |
Jun 03, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Carme Camps |
| Organization name |
Wellcome Trust Centre for Human Genetics/ U. of Oxford
|
| Street address |
Roosevelt Drive
|
| City |
Oxford |
| ZIP/Postal code |
OX37BN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE47534 |
Integrated analysis of microRNA and mRNA expression and association with HIF binding in MCF-7 cells under hypoxia |
| GSE47602 |
Integrated analysis of microRNA and mRNA expression and association with HIF binding in MCF-7 cells under hypoxia (miRNA-Seq) |
|
| Relations |
| BioSample |
SAMN02189822 |
| SRA |
SRX290632 |
