RNA-seq WT-1
RNA-seq library preparation was performed as previously described. Total RNA was isolated and analyzed on the Agilent Tape station for RNA integrity numbers (RINs) before library preparation. RNA-seq libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (NEB, Cat. No. E7775) according to the manufacturers protocol. mRNA was isolated using poly-T oligos conjugated to magnetic beads and then fragmented and reverse-transcribed to first strand cDNA. During the synthesis of the second strand, deoxyuridine triphosphates (dUTPs) were included and consequently were not amplified. Double strand cDNA was purified by SPRIselect Beasds (Beckman Coulter). cDNA underwent end prep reaction, ligation with indexed adaptors, purification, and PCR amplification. PCR reaction was purified by SPRIselect Beasds (Beckman Coulter).