Bulk RNA-seq of ESCC PDOX
cDNA libraries were prepared by KAPA mRNA HyperPrep Kit. One hundred nanograms of total RNA was used as starting material. Manufacturers protocol was followed. In brief, Poly-A containing mRNA was collected by using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented to 200  300 bp by incubating at 94oC for 6 min in the presence of magnesium ions. The fragmented mRNA was then applied as template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent, 3 adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 15 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing.