GSM5509132: ND_6232; Homo sapiens; RNA-Seq
PicoPure RNA extraction kit (Thermofisher Scientific, cat# 12204-01) with genomic DNA removal using recombinant DNAse1 (Worthington Biochemical Corporation, cat# LS006361) Agilent 2100 Bioanalyzer (Picotape, Agilent Technologies) A sample of 6 ng RNA was used to extract mRNA with 15μl of NEBNext Magnetic Oligo d(T) and fragmented in NEBNext First Strand Synthesis Buffer by heating at 94°C for 8 minutes in a 96-well plate. First strand cDNA synthesis was performed using reverse transcriptase and random primers. Synthesis of double-stranded (ds) cDNA was performed using the second strand master mix provided in the kit. The resulting ds cDNA was end-repaired, dA-tailed, and ligated with NEBNext adaptors. The library was enriched by PCR amplification and purified by Agencourt AMPure beads (Beckman Coulter). Barcoded libraries were sized on the Bioanalyzer and quantitated by QUBIT. The library's functionality was validated by quantitative reverse transcription PCR (qRT-PCR), using the KAPA library quantification kit (Kapa Biosystems).