|
| Status |
Public on Jul 24, 2013 |
| Title |
Proliferatives_rep3_RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
Proliferatives_RNAseq
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: WI38
cell type: primary fibroblasts
status: Proliferative
genotype/variation: infected with pBABE-puro-empty
|
| Treatment protocol |
Infection by retroviral-mediated gene transfer with either pBABE-puro-H-RasG12V or empty vector were performed as previously described (Bischof et al., 2006)
|
| Growth protocol |
Culturing of human diploid WI38 fibroblasts (ATCC) was performed at physiological oxygen concentration of 3% as previously described (Bischof et al., 2006)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using RNeasy RNA isolation kit (Quiagen) 5 days post-infection
libraries were prepared using mRNA-seq sample prep kit (#RS-100-0801, Illumina Inc.) following the manufacturer's protocol with some modifications. Briefly, mRNA was purified from 2 µg of total RNA using oligo-dT magnetic beads and fragmented using divalent cations at 95°C for 5 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers and SuperScript II reverse transcriptase (# 18064-014, Invitrogen). This was followed by second strand cDNA synthesis using Polymerase I and RNase H. The double stranded cDNA fragments were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were separated by 2% agarose gel electrophoresis and ~200-250bp fragments were excised and purified using QIAquick Gel Extraction Kit (Qiagen) and then enriched by PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] x 13 cycles; 5 min at 72°C). After PCR amplification, surplus PCR primers were removed by purification using Agencourt AMPure XP beads (#A63881, Beckman). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent). The libraries were loaded in the flowcell at 6pM concentration and clusters were generated using the Cbot and sequenced on the Illumina Genome Analyzer IIx as single-end 54 base reads following Illumina’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.7
sequence reads mapped to reference genome hg19 using Tophat (Trapnell et al., 2009). Quantification of gene expression was done using Cufflinks (Trapnell et al., 2010) and annotations from Ensembl release 57. For each transcript the resulting FPKM were converted into raw read counts and these counts were added for each gene locus. Data normalization was performed as described by Anders et al. (Anders and Huber, 2010) and implemented in the DESeq Bioconductor package.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include Ensembl release 57 human gene id, RPKM values for each Sample.
|
| |
|
| Submission date |
Nov 09, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Tao YE |
| Organization name |
IGBMC (CNRS/INSERM/UDS)
|
| Street address |
1 rue Laurent Fries
|
| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE42212 |
Quantitative analysis of proliferative and Ras-induced senescent human primary fibroblasts transcriptomes |
| GSE42213 |
SUMO is an Integral and Instructive Component of Chromatin in Cell Growth and Senescence |
|
| Relations |
| SRA |
SRX204183 |
| BioSample |
SAMN01805536 |
