GSM2477706: E-1056; Homo sapiens; miRNA-Seq
500 ul of human serum were submitted for each sample. SMall RNA from each serum sample was isolated using the miRCURYTM RNA isolation kit (Exiqon, Vedbæk, Denmark) according to the manufacturer’s protocol optimized for serum. The quality of the isolated RNA was checked using qPCR. Total RNA was converted into microRNA NGS libraries using the NEBNEXT library generation kit (New England Biolabs Inc., Ipswich, MA) 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 indices. After 18 cycles of pre-PCR the libraries were purified on QiaQuick columns and the insert efficiency evaluated by a Bioanalyzer 2100 instrument on a high sensitivity DNA chip (Agilent Inc., Lexington, MA). The microRNA cDNA libraries were size fractionated on a LabChip XT (PerkinElmer Waltham, MA) and a band representing adaptors and 15-40 bp insert excised using the manufacturer’s instructions. Samples were then quantified using qPCR and concentration standards. Based on the quality of the inserts and the concentration measurements, the libraries were pooled in equimolar concentrations (all concentrations of libraries to be pooled were of the same concentration). The library pools were finally quantified again with qPCR and the optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing using v3 sequencing methodology according to the manufacturer instructions (Illumina Inc., Dedham, MA). Samples were sequenced on the Illumina NextSeq 500 system (Illumina Inc., Dedham, MA) using a single-end read length of 50 nucleotides at an average of 10 million reads per sample.
GEO Accession
GSM2477706