GSM8446981: totalRNA_siLSM14A_NT_REP1; Homo sapiens; RNA-Seq
RNA was isolated using the TRIzol-chloroform method and treated with DNase I (TURBO DNA-free; Invitrogen™, #AM1907) according to the manufacturer's instructions. After DNase treatment, RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific™), and 1 μg total RNA was used to synthesize cDNA using SuperScript III Reverse Transcriptase (200 U/μL; Invitrogen™, #18080044) and random hexamer primers (Invitrogen™, #SO142), according to the manufacturer's instructions. RNA sequencing libraries were prepared from 500ng to 1µg of total RNA or mRNA-enriched from heavy polysome fractions using the Illumina TruSeq Stranded mRNA Library preparation kit which allows to perform a strand specific sequencing. Nanodrop spectrophotometer was used to assess purity of RNA based on absorbance ratios (260/280 and 260/230) and BioAnalyzer for RNA integrity (RIN>9). A first step of polyA+ selection using magnetic beads is done to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing followed by ligation of TruSeq indexed adapters. PCR amplification was finally achieved to generate the final barcoded cDNA libraries. The libraries were equimolarly pooled and subjected to qPCR quantification using the KAPA library quantification kit (Roche).