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| Status |
Public on Apr 13, 2015 |
| Title |
SLKK_polyA_mRNAseq_rep3 [mRNA] |
| Sample type |
SRA |
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| Source name |
SLKK cells
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| Organisms |
Homo sapiens; Human gammaherpesvirus 8 |
| Characteristics |
infection status: Chronically KSHV-infected
cell line: SLKK cells
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| Growth protocol |
Human KS-derived SLK and SLKK cells (also known as SLK+rKSHV.219) (Herndier et al., 1994; Stürzl, Gaus, Dirks, Ganem, & Jochmann, 2013; Jeffrey Vieira & O’Hearn, 2004; Ziegelbauer, Sullivan, & Ganem, 2009) were 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 10mg/mL puromycin to maintain the viral episome. Experiments were done in triplicate independent cell cultures maintained at 37°C in humidified 5% CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from cells using miRVana miRNA isolation kit (Ambion, Life Technologies) according to manufacturer instructions. RNA abundance and integrity were determined after isolation using a Nanodrop-ND-1000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer, respectively.
For mRNA-Seq: Total RNA of samples used for small RNA-Sequencing 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) were sequenced using the Illumina HiSeq platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Total RNA enriched for polyadenylated RNA
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| 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
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| Submission date |
Oct 29, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Coralie Viollet |
| Organization name |
NIH/NCI
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| Street address |
10 Center Dr
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20814 |
| Country |
USA |
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| Platform ID |
GPL19367 |
| Series (2) |
| GSE62829 |
Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of miRNA-mRNA Target Pairs in KSHV-Infected Cells [mRNA-Seq] |
| GSE62830 |
Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of miRNA-mRNA Target Pairs in KSHV-Infected Cells |
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| Relations |
| BioSample |
SAMN03152227 |
| SRA |
SRX747074 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1534027_SLKK_mRNAseq_rep3.bedGraph.gz |
178.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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