TRIzol (Invitrogen), was used to  extract total RNA from 30 primary tumors. The RNA samples were cleaned using RNeasy cleanup (Qiagen), respectively. RNA was quantified on a NanoDrop Microvolume  Spectrophotometer and quality was assessed by Bioanalyzer (Agilent). The total RNA was extracted from 30 samples were 300 ng were processed using Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, CA, USA, Catalogue # R3000) to generate RNAseq libraries.