|
| Status |
Public on Dec 21, 2016 |
| Title |
SLKK_hypoxia_polyA_mRNAseq_rep3 [mRNA] |
| Sample type |
SRA |
| |
|
| Source name |
SLKK cells
|
| Organism |
Homo sapiens |
| Characteristics |
infection status: Chronically KSHV-infected (Human herpesvirus 8)
oxygen concentration: Hypoxia (1% O2 for 24hrs)
cell type: SLKK cells
|
| Treatment protocol |
Exposure of cell cultures to 1% oxygen was undertaken in an InVivo2 Hypoxia Work Station (Ruskinn Technology, UK). This was undertaken in parallel with cells maintained in normoxic conditions (21% O2). All cells were harvested for protein and RNA 24hrs post-treatment. All experiments were done at least in triplicate from independent cell cultures.
|
| Growth protocol |
Human KS-derived SLK and SLKK cells (also known as SLK+rKSHV.219) were a gift from Dr. Don Ganem (UCSF, CA). They were expanded on receipt, frozen in liquid nitrogen, and stored in a cryogenic tank until used in the experiments described hereafter. Cells were thawed and maintained in Dulbecco’s Modified Eagle medium supplemented with 10% v/v fetal bovine serum and 1% Penicillin/streptomycin/glutamine solution. Additionally, KSHV-positive SLKK cells were periodically grown under selection with 10 μg/mL puromycin to maintain the viral episome.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using miRVana miRNA isolation kit according to manufacturer’s instructions. RNA abundance and integrity were determined after isolation using a Nanodrop-ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Only samples of total RNA with an RNA integrity number (RIN) >9 were further used for RNA-sequencing and small RNA-sequencing. All samples were stored at -80°C.
For mRNA-Seq: Total RNA was treated with Turbo DNA-free DNase I and Dynabeads to deplete samples from the residual DNA and to isolate the polyadenylated mRNA transcriptome, respectively. PolyA+ RNA libraries were then prepared with the ScriptSeq v2 RNA-Seq kit (Epicentre). The final concentration and size distribution of the RNA libraries were measured by using a Nanodrop-ND-1000 spectrophotometer, and by running a DNA 100 chip on an Agilent 2100 Bioanalyzer. Finally, a total of 6 polyadenylated mRNA libraries (3 SLK and 3 SLKK samples in hypoxia) were sequenced using the Illumina HiSeq platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Total RNA containing short RNA fraction; DNase treated; polyA enriched fraction
|
| Data processing |
For mRNA sequencing: Adapter sequences were trimmed using the Fastx toolkit. Reads were aligned against two target genomes, human (hg19) and KSHV (Genbank accession number NC_009333.1) using TopHat (Trapnell, Pachter, & Salzberg, 2009) to generate spliced alignments. Transcripts were assembled using Cufflinks and Cuffdiff (Trapnell et al., 2010) in order to reveal differentially expressed genes. Significant mRNA fold change was determined by an adjusted p-value lower than 0.05 based on the Benjamini and Hochberg multiple testing correction.
Genome_build: Human genome (hg19) and KSHV genome (Genbank accession number NC_009333.1)
Supplementary_files_format_and_content: bedGraph files were generated using BEDtools
Supplementary_files_format_and_content: Cufflinks ouput
Supplementary_files_format_and_content: Cuffdiff output
|
| |
|
| Submission date |
Mar 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Coralie Viollet |
| Organization name |
NIH/NCI
|
| Street address |
10 Center Dr
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20814 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE79029 |
KSHV Infection Mimics the Hypoxic Response Based on Next-Generation Sequencing [mRNA-Seq] |
| GSE79032 |
RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature |
|
| Relations |
| BioSample |
SAMN04543019 |
| SRA |
SRX1620845 |
