Illumina Small RNA Sequencing from Serum

Small RNA sample preparation was performed using NEBNext^® Multiplex Small RNA Library prep set for Illumina (Set 1) according to the manufacturer's instructions. Briefly, 3’ and 5’ adaptors were sequentially ligated to serum total RNA, using 6μl input RNA per sample. A mix of ten different calibrator oligoribonucleotides (0.25μl) with known sequence and concentration were added in the 3’ligation step and used as internal standards as described by Hafner and colleagues [45 (link)]. The following steps included reverse transcription of the ligated fragments, amplification by PCR for 13 cycles using Index primers from NEBNext^® Multiplex Small RNA Library prep set for Illumina Set 1 and Set 2, and gel purification. Quality controls of the cDNA libraries were measured using High Sensitivity DNA assay on 2100 Bioanalyzer. The miRNA fragments were sequenced on the Illumina HiSeq system using 50 base pair single read at the Genomics Core Facility (GCF) at the Norwegian University of Science and Technology in Trondheim, Norway.