|
| Status |
Public on Jun 18, 2012 |
| Title |
MCF10A proliferative |
| Sample type |
SRA |
| |
|
| Source name |
MCF10A breast epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: MCF10A breast epithelial cells
growth stage: proliferative
rna subtype: polyA RNA
|
| Treatment protocol |
BJ-EHT/p53kd/p16kd/RASGV12ER cells expressing short hairpin (sh)RNA constructs targeting p53 and p16INK4A and 4-hydroxy-tamoxifen (4-OHT) inducible oncogenic HRASG12V were cultured for 3 days in the presence of 100 nM 4-OHT to transform the cells. To transform the MCF10A cells we transduced them with a retroviral vector expressing RASG12VER, and cultured them for 2 and 8 days in the presence of 100 nM 4-OHT
|
| Growth protocol |
Primary BJ foreskin fibroblasts containing expression constructs for the ecotropic receptor and the human telomerase gene (hTERT), BJ-EHT, were either maintained in a cycling state or contact inhibited by growing them to confluence in DMEM (Gibco) supplemented with 10% FCS and antibiotics. Non-transformed mammary epithelial MCF10A cells were either cultured in DMEM:F12 Ham's medium (Sigma) 1:1, supplemented with 10% fetal calf serum (Gibco), insulin (10 μg ml−1; Sigma), hydrocortisone (0.5 μg ml−1) and epidermal growth factor (20 ng ml−1; Peprotech) or serum starved with DMEM:F12 containing no supplements for 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit from Qiagen. mRNA was isolated with the Oligotex mRNA Kit from Qiagen.
Total RNA was extracted from cells. mRNA was then isolated, heat fragmented and the fragmented mRNA was converted to single-stranded cDNA using SuperScript III reverse transcriptase and P7-t25-vn oligo-dT primer. This was followed by second-strand cDNA synthesis and end-repair using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. After purification, a P5-splinkerette was ligated and the splikerette-ligated cDNA was size-selected for approximately 220 bp long cDNA fragments using the E-Gel iBase Power System from Invitrogen. The final 3'Seq libraries were then generated by PCR amplification of the linker-ligated cDNA with P5 and P7 primers.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Illumina GA-II, HiSeq 2000
|
| Data processing |
Reads were aligned to the human genome (hg18) using bowtie. Wig files which summarize read coverage at each genomic ccordinate (hg18) were generated using a perl script and transcripts' genomic coordinates downloaded from the USCS browser related Tables.
|
| |
|
| Submission date |
Nov 09, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Ran Elkon |
| Organization name |
Tel Aviv University
|
| Department |
Human Genetics
|
| Street address |
Ramat Aviv
|
| City |
Tel Aviv |
| ZIP/Postal code |
69494 |
| Country |
Israel |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE33592 |
Wide-scale analysis of alternative polyadenylation (APA) associated with proliferation and transformation using 3'-Seq |
|
| Relations |
| SRA |
SRX105304 |
| BioSample |
SAMN00752530 |
