GSM9321146: PPB_miRNAseq_rep1; Homo sapiens; miRNA-Seq
Total RNA was isolated from fresh frozen specimens with TRIzol reagent according to the manufacturer's instructions (Invitrogen). Samples in OCT were cut in 20 µm-thick sections and 20-30 sections per specimen were used for extraction with TRIzol (Invitrogen) according to manufacturer's instructions. The NEBNext multiplex small RNA library prep ((New Englands Biolabs Inc., USA) was used to prepare miRNA sequencing libraries, according to manufacturer's instructions. Libraries were prepared using 500ng of total RNA. Briefly, RNA 3' adapter was first ligated to the 3' end of miRNA followed by the ligation of RNA 5' adapter to the 5'end of miRNA-3' adaptors, which were further used for the reverse transcription step. Following the reverse transcription step, an amplification of 14 cycles was performed to incorporate specific indexes for multiplexing. After a purification step with GenElutePCR clean-up kit (Sigma-Aldrich, USA), the appropriate range of cDNA fragments was extracted on a 3% gel using a Pippin Prep instrument (Sage Science, USA). Llibrary quantification was done on a QBit 3.0 fluorometer (ThermoFisher Scientific, Canada). miRNA libraries were pooled together in equimolar ratio and the quality was examined with a DNA screentape D1000 HS on a TapeStation2200 (Agilent technologies, USA). Subsequently, miRNA librairies with unique index were sequenced for paired-end 100pb sequencing on an Illumina Novaseq 6000.