|
| Status |
Public on Dec 06, 2013 |
| Title |
BCT32_mRNA |
| Sample type |
SRA |
| |
|
| Source name |
Breast tumor
|
| Organism |
Homo sapiens |
| Characteristics |
tissue type: HER2+ Breast Tumor
|
| Treatment protocol |
Total RNA extraction was performed using Exiqon's miRCURY RNA Isolation Kit. Long-read mRNA-seq cDNA libraries were prepared from 1ug of total RNA using a modification of the Illumina mRNA-seq protocol. Briefly, mRNA was resolved using poly-dT oligonucleotides attached to magnetic beads, fragmented using divalent cations under elevated temperatures, and converted to cDNA using random primers. After conversion of the cleaved fragments into cDNA, the cDNA underwent blunt end repair, addition of an 'A' base to the 3' blunt ends, and ligation of adapter molecules which will be used for PCR amplification, bridge amplification, and sequencing. The cDNA library was resolved by gel electrophoresis using conventional Illumina protocols except that we cut from the gel those cDNA fragments in the range of 300-400bp. The increased fragment length is necessary to accommodate paired end sequence analysis. The gel purified cDNA fragments were amplified by PCR and sequenced using the Illumina Cluster Station and Genome Analyzer. Paired-end sequence analysis (51 cycles/end) was carried out using sequencing primers that correspond to either end of the bridge-amplified cDNA fragments so as to obtain 50nt of sequence from either end of every cDNA fragment.
|
| Growth protocol |
All the tumors and cell lines were grown under conditions recommended by ATCC and RNA and DNA were extracted from mid log phase populations of low passage number cultures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the human (NCBI 37 build) genomes using BWA and tophat 1.3 alignment. All reads mapping with two or fewer mismatches were retained and read starts were summed .
Raw Gene Count: Gene counts were obtained using HTSeq
Variant callng is done using eSNV Detect Pipeline
Genome_build: hg19
Supplementary_files_format_and_content: Text file with raw gene counts for all the samples(tab seperated)
|
| |
|
| Submission date |
Mar 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Krishna R Kalari |
| E-mail(s) |
kalari.krishna@mayo.edu
|
| Organization name |
Mayo Clinic
|
| Department |
Department of Biomedical Statistics and Informatics
|
| Lab |
Dr Thompson-CancerBiology
|
| Street address |
200 1st ST SW
|
| City |
Rochester |
| State/province |
MN |
| ZIP/Postal code |
55901 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE45419 |
An Integrated Model of the Transcriptome Landscape of HER2-Positive Breast Cancer |
|
| Relations |
| SRA |
SRX254200 |
| BioSample |
SAMN01985676 |
