GSM4977298: RNA-AST2-11; Homo sapiens; RNA-Seq
Total RNA for each sample was isolated by using TRIzol. RNA concentration was determined using the Qubit® 2.0 Fluorometer (ThermoFisher/Invitrogen, Grand Island, NY), and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). RNA samples with a measured 28S/18S > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction. RNA-seq library were constructed using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations. Approximately 125 ng of total RNA was used for mRNA isolation using the NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490). RNA library construction was then performed with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, catalog #E7760) according to the manufacturer's user guide. Barcoded libraries were sized on the bioanalyzer and quantitated by QUBIT. Six individually prepared libraries (n=3 for AST and n=3 for NST) were pooled by equimolar and sequenced by Illumina HiSeq 3000 2X100 cycles run (Illumina Inc., CA, U.S.A). RNA library construction and sequencing were performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression & Genotyping Core, University of Florida (UF).