|
| Status |
Public on Mar 29, 2012 |
| Title |
DCIS |
| Sample type |
SRA |
| |
|
| Source name |
ductal carcinoma in situ cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: DCIS
disease status: ductal carcinoma in situ
Stage: premalignant stage
|
| Growth protocol |
All the experiments in the present study were conducted using in vitro three dimensional (3D) organotypic culture models with reconstituted basement membrane (rBM). In the 3D rBM overlay protocol, single cells were seeded onto culture dishes previously coated with polymerized rBM (Cultrex®) and were overlaid with media containing 2% rBM when the cells had attached.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We used Trizol to extract the RNA from our samples. Libraries of template molecules for high throughput DNA sequencing were prepared using Illumina mRNA Sequencing Sample Preparation kit. First, using Sera-Mag oligo (dT) beads, mRNA molecules were purified from the total RNA. Then the purified mRNA samples were fragmented under elevated temperature conditions. Subsequently, first strand cDNA synthesis was done from the cleaved RNA fragments using reverse transcriptase and random primers. This was followed by second strand synthesis using DNA polymerase I and RNaseH (using SuperScript II from Invitrogen). These cDNA fragments were then subjected to repair end process with T4 DNA polymerase and Klenow. The 3’ ends were then adenylated to prepare for adapter ligation. After paired end ligation of adapters, the samples were purified on 1.2% agarose recovery FlashGelTM. The library of products of desired size (150-200bp) was then selected for further enrichment with 15 cycles of polymerase chain reaction (PCR) amplification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Sequence reads were obtained and mapped to the human (March, 2006) genomes using novoalign with default parameters. Only the reads that map to the unique genome positions were considered.
Genome Build:
DCIS_2_novo.bed: hg18
DCIS_3_novo.bed: hg18
|
| |
|
| Submission date |
Mar 27, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
RAYMOND R MATTINGLY |
| E-mail(s) |
ae8291@wayne.edu
|
| Phone |
313-577-6022
|
| Organization name |
Wayne State University
|
| Department |
Pharmacology
|
| Street address |
540 East Canfield
|
| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48201 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE36863 |
DEEP SEQUENCING OF MODELS OF BREAST DUCTAL CARCINOMA IN SITU REVEALS ALDH5A1 AS A NOVEL POTENTIAL THERAPEUTIC TARGET |
|
| Relations |
| SRA |
SRX131949 |
| BioSample |
SAMN00840776 |
