GSM8008167: wild-type, rep 1; Homo sapiens; RNA-Seq
The total RNA of each sample was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, and the residual DNA was removed using the RapidOut DNA Removal Kit (Thermo Fisher Scientific, USA). To prepare the library, 1 μg of total RNA was used. Poly(A) mRNA isolation was performed using Oligo(dT) beads, and mRNA fragmentation was carried out using divalent cations and high temperature. Random primers were used for priming, and first and second-strand cDNA synthesis was performed. The double-stranded cDNA was then purified, and both ends were repaired and dA-tailed in a single reaction, followed by T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was performed using DNA Clean Beads. Each sample was amplified by PCR using P5 and P7 primers, and the PCR products were validated. The libraries with different indexes were then multiplexed, and the Illumina NovaSeq 6000 instrument was used for sequencing in a 2 ×150 paired-end configuration following the manufacturer's instructions.