|
| Status |
Public on Aug 06, 2013 |
| Title |
IFN-g stimulated HT-29 cells run at 25M reads |
| Sample type |
SRA |
| |
|
| Source name |
colon
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: colon adenocarcinoma
cell line: HT-29
treatment: IFN-g
sample type: 25 M reads
|
| Treatment protocol |
HT-29 cells were treated with 200 U ml-1 IFN-γ (Roche) for 24 hr.
|
| Growth protocol |
HT-29 cells were cultured in McCoy's 5A medium with 10% FBS and 5% CO2. MCF10A cells were cultured in DMEM/F12 medium with 5% horse serum, 20 ng/ml EGF, 500 ng/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 µg/ml insulin. MDA-MB-436 cells were cultured in L-15 medium with 10% FBS without CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocols.
Each cDNA library was prepared with the NEBNext first-strand synthesis, second-strand synthesis, end repair, dA tailing, and quick ligation modules (New England Biolabs). Libraries were indexed with standard Illumina-type adapters and sequenced on an Illumina HiSeq 2000 using version 3 reagents that generate 180–200M reads per lane. Samples were 50-bp paired-end sequenced in duplicate at 25M and 50M or 100M reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
RNA-seq reads were chastity filtered and assessed for overall quality with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Sequenced reads were mapped using STAR 2.2.0 against the human genome build hg19.
Reads that map to each gene were counted with HTSeq (http://www-huber.embl.de/users/anders/HTSeq/) under the union set, whereby reads that do not completely overlap a gene are still counted. The counts were normalized by the library size (read depth).
RPKM calculations were performed by normalizing to the median transcript length for each gene and the total library size of each sample.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include normalized count in the first column and RPKM value in the second column for each sample
|
| |
|
| Submission date |
Mar 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Byong Ha Kang |
| E-mail(s) |
bhk3j@virginia.edu
|
| Organization name |
University of Virginia
|
| Department |
Department of Biomedical Engineering
|
| Lab |
Janes Lab
|
| Street address |
415 Lane Road, MR5-Rm. 2225
|
| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22908 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE45258 |
RNA-seq analysis of HT-29, MCF10A, and MDA-MB-436 cells |
|
| Relations |
| SRA |
SRX251971 |
| BioSample |
SAMN01983941 |
