GSM5416602: LCL65JW; Homo sapiens; miRNA-Seq
Cells were split at ½ dilution approximately 24h before being collected for RNA extraction with TRI-Reagent (Sigma Aldrich) following the manufacturer's instructions. Small RNA-Seq libraries were generated from 1000 ng of total RNA using TruSeq Small RNA Library Prep Kit (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, in the first step, RNA adapters were sequentially ligated to each end of the RNA, first the 3' RNA adapter that is specifically modified to target microRNAs and other small RNAs, then the 5' RNA adapter. Small RNA ligated with 3' and 5' adapters were reverse transcribed and PCR amplified (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 15 sec at 72°C] x 13 cycles; 10 min at 72°C) to create cDNA constructs. Amplified cDNA constructs of 20 to 40 nt were selectively isolated by acrylamide gel purification followed by ethanol precipitation. The final cDNA libraries were checked for quality and quantified using capillary electrophoresis and sequenced on the Illumina HiSeq 4000 at the Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) GenomEast sequencing platform.