GSM2671476: RH30 WT rep3 (WT3); Homo sapiens; miRNA-Seq
Total RNA was isolated using the mirVana miRNA Isolation Kit (Ambion). Library preparation, next generation sequencing and analysis of data were performed at Exiqon Services, Denmark. A total of 1 ug of total RNA was converted into microRNA NGS libraries using NEBNEXT library generation kit (New England Biolabs Inc) according to the manufacturer’s instructions. Each individual RNA sample had adaptors ligated to its 3’ and 5’ ends and converted into cDNA. Then the cDNA was pre-amplified with specific primers containing sample specific indexes. After 15 cycle pre-PCR the libraries were purified on QiaQuick columns and the insert efficiency eavaluated by Bioanalyzer 2100 instrument on high sensitivity DNA chip (Agilent Inc.) The microRNA cDNA libraries were size fractionated on a LabChip XT (Caliper Inc.) and a band representing adaptors and 15-40 bp insert excised using manufacturer’s instructions. Samples were then quantified using qPCR and concentration standards. Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar concentrations (all concentrations of libraries to be pooled are of the same concentration). The library pools were finally quantified again with qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing using v2 sequencing methodology according to the manufacturer instructions (Illumina Inc.). MicroRNA sequencing. Samples were sequenced on the Illumina NextSeq 500 system. The system uses quality score binning enabling a more compact storage of raw sequences. Using only 8 levels (Levels: No call, 6, 15, 22, 27, 33, 37, 50) of quality method has been tested and found to virtually loss-less. Average number of reads per sample was 7.5mio. Number of sequencing cycles (read length) was 50 nt , single-end read.
GEO Accession
GSM2671476