GSM5734414: JW_04: ShG6PD_01; Homo sapiens; RNA-Seq
Total RNA was isolated from cultured cells using a RNeasy kit (Cat. 74106, Qiagen) according to the manufacturer's instructions. RNA was quantified using a NanoDrop 100 spectrophotometer (Thermofisher). Quality of the RNA was assessed by measuring the ratios of absorbance (A260/A280 and A260/A230); ratios greater than 1.8 were considered suitable for further analysis. cDNA synthesis RNA was DNase I treated, cleaned and concentrated according to the manufacturer's instructions using Zymo RNA Clean and Concentrator -5 (R1013, Zymo). mRNA was enriched from total RNA according to the manufacturer's instructions using a NEBnext poly(A) mRNA magnetic isolation module. Firstly mRNA was isolated and fragmented from total RNA using oligo dT beads and a magnetic rack. Beads were mixed with 1μg RNA and then washed twice. Samples were then heated to 65˚C for 5mins before being washed. Tris buffer was added and samples were heated once again to 80˚C and held at 25˚C to perform the first elution of the mRNA from the beads. Beads were subsequently washed and mRNA was eluted and fragmented with the addition of first strand synthesis reaction buffer and random primer mix. Samples were heated to 94˚C for 15 min and held at 4˚C Sequencing libraries were prepared according to the manufacturer's recommendations using the NEBNext Ultra II RNA Library Prep kit (E7770S, NEB). Briefly, first and second strand cDNA synthesis was performed using mRNA isolated as above. Double strand cDNA was purified using NEBNext sample purification beads, and DNA was eluted in 0.1X TE buffer. End prep reaction and adaptor ligation was performed and purification was done using sample purification beads. RNA and DNA quantification was performed using HS RNA and HS DNA assay kits (Q32852, Q32851, Invitrogen) Qubit fluorometer (Life Tech) according to the manufacturer's instructions. RNA and DNA quality was determined using High Sensitivity RNA Screentape and D1000 High Sensitivity DNA Screentape respectively on Agilent 4200 tapestation system according to the manufacturer's instructions. Single-indexed and multiplexed samples were run on an Illumina Next Seq 500 sequencer using a NextSeq 500 v2 kit (150 cycles) (FC-404-2005) for paired-end sequencing (75 bases x 2). Results from RNA-Seq were aligned to the human genome assembly version 38 (hg38 or GRCh38). At least 14 million aligned reads were obtained per sample