GSM4848823: A2T_S6; Homo sapiens; RNA-Seq
Total RNA was purified using the RNeasy plus MiniKit (Qiagen) and the integrity of RNA was assessed using the RNA pico chip on Bioanalyzer 2100 (Agilent) and quantified with Qubit Fluorometer (Thermo Fisher). RNA Integrity Numbers (RINs) were used to evaluate the integrity of the RNA samples with > 7.0 considered intact and selected these samples for RNA library prep. The RNA libraries were prepared and sequenced at the University of Houston Seq-N-Edit Core per standard protocols. RNA libraries were prepared with SMARTer Stranded Total RNA-Seq Kit v1 - Pico Input Mammalian Kit (Takara) using 10ng input RNA. RNA was fragmented, reverse transcribed into cDNA with random primers for the first strand and LNA-TSO switching oligo for the second stranded to give stranded information. Illumina adaptors were ligated to the cDNA at the 5' and 3' ends and rRNA was cleaved with ZapR enzyme. The uncleaved cDNA was amplified and size selection for libraries was performed using SPRIselect beads (Beckman Coulter). The purity of the libraries was analyzed using the DNA high sensitivity chip on Bioanalyzer 2100 (Agilent) and sequences were ~400bp in size. The indexed libraries were pooled and sequenced using NextSeq 500 (Illumina); generating ~20million 2×76 bp paired-end reads per samples.