Bulk RNA-seq of in vitro NE/ESCC cell lines and hNEEO/ESCCPDO/EACPDO organoid cultures
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.