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| Status |
Public on Apr 13, 2015 |
| Title |
SLKK_microRNAseq_rep2 [microRNA] |
| 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 iSLK and iSLK.219/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 |
total 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 microRNA-Seq: Small RNA libraries were constructed using the Illumina TruSeq RNA Sample Prep kit. Briefly, after running 2μg of total RNA in a 15% urea-TBE gel for 1hr at 200V, the 20 to 30 nucleotide RNA fraction was excised and eluted in 0.3M NaCl. Following separation of the elute from the gel debris using a Spin-X-column, the small RNA samples were precipitated in 100% ethanol and 1mg.mL-1 glycogen, incubated at -80°C for 30 minutes, centrifuged at 14,000rpm for 25 minutes, washed with 75% ethanol, air dried and resuspended in RNAse-free water. Illumina TruSeq libraries were then prepared according to the manufacturer’s protocol and the final RNA library concentration was measured by a Qubit 2.0 fluorometer using Qubit dcDNA HS Assay kit. We verified the size of the products contained in the libraries using a high sensitivity DNA chip and an Agilent 2100 Bioanalyzer. Finally, a total of 6 miRNA libraries (3 SLK and 3 SLKK samples) were sequenced using the Illumina HiSeq platform.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Total RNA containing short RNA fraction
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| Data processing |
For small RNA sequencing: Reads were aligned against known miRNAs from miRbase (version 19.0) using the software package SHRIMP (Rumble et al., 2009) (http://compbio.cs.toronto.edu/shrimp/README) with miRNA specific settings (-o 1 -n 2 -r 30% -h 50% --local -Q --qv-offset 32 --sam). To process paired-end sequencing, reads were aligned separately covering the mature miRNA both on the forward and reverse read and the obtained number of matches were averaged. The match counts were normalized and tested for differential expression using the edgeR package (McCarthy, Chen, & Smyth, 2012) with default settings.
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) |
| GSE62828 |
Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of miRNA-mRNA Target Pairs in KSHV-Infected Cells [microRNA-Seq] |
| GSE62830 |
Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of miRNA-mRNA Target Pairs in KSHV-Infected Cells |
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| Relations |
| Reanalyzed by |
GSE79030 |
| BioSample |
SAMN03152222 |
| SRA |
SRX747064 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1534020_SLKK_microRNAseq_rep2.bedGraph.gz |
526.4 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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