GSM1534020: SLKK_microRNAseq_rep2 [microRNA]; Homo sapiens; Human gammaherpesvirus 8; miRNA-Seq
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.
GEO Accession
GSM1534020