GSM4969969: OS-LM1-sgHNF1B_2_REP_4; Homo sapiens; RNA-Seq
RNA was extracted from 80% confluent cells 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 Bioanalyzer 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.