GSM4968791: ccRCC_24H_sample_3; Homo sapiens; RNA-Seq
RNA was extracted from fresh frozen tumours using the RNeasy Mini Kit (Qiagen 74104) following the manufacturer's recommendations. RNA quality was assessed with the RNA Nano 6000 kit (Agilent 5067-1511) and Agilent Bionalyzer 2100 instrument. Purified RNA (1µg) was used to prepare Illumina multiplexed sequencing libraries. Libraries were prepared following the KAPA Stranded mRNA-seq Kit (KR0960) Illumina platforms sample preparation protocol (v3.15). Briefly, poly(A) RNA was captured with magnetic oligo-dT beads and heat fragmented (94°C) to a size of 200-300bp. Fragmented RNA was used for 1st strand cDNA synthesis, generating cDNA-RNA hybrids which were used for 2nd strand synthesis and marking to obtain dscNDA marked with dUTP. Newly synthesized fragments were end-repaired and A-tailed to add dAMP to the 3'-end of dscDNA fragments. These 3'-dA-tailed library fragments were then ligated with 3'-dTMP adapters. After adapter ligation, libraries were cleaned up using the Agencourt AMPure XP reagent (A63880) according to manufacturer's recommendations. Purified, adapter-ligated DNA was amplified for 15 cycles. PCR libraries were pooled in equimolar concentrations and sequenced.