GSM5381341: 4701_S7_IO_0014_DMSO; Homo sapiens; RNA-Seq
Total cellular RNA was extracted from TMCs using RNAeasy Mini kit (Qiagen). Total RNA quality and quantity was assessed using the Agilent Bionalyzer 2100 and the Qubit fluorometer, respectively. Poly(A) mRNA capture and construction of stranded mRNA-seq libraries from intact total RNA (RIN >7) was achieved using the commercially available KAPA Stranded mRNA-Seq Kit. In brief, mRNA transcripts were first captured from 200ng of total RNA using magnetic oligo-dT beads, fragmented using heat and magnesium, and reverse transcribed to produce dscDNA. Illumina sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. Libraries were indexed using dual molecular indices allowing for multiple libraries to be pooled and sequenced on the same sequencing lane on a HiSeq 4000 Illumina sequencing platform. Before pooling and sequencing, fragment length distribution and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies). All libraries were then pooled in equimolar ratio and sequenced. Multiplexing 8 libraries on one lane of an Illumina HiSeq 4000 flow cell yielded about 43 million 50bp sequences per sample. Once generated, sequence data was de-multiplexed and Fastq files generated using Bcl2Fastq conversion software provided by Illumina.