title: RNA-Seq of DXNEN-YZ-009TB
design: Total RNA was isolated from primary patient tumors and paired tumor organoids using trizolAmbion/InvitrogenUSAand received strict quality control through Agilent 2100 Bioanalyzer. The acquisition of mRNA is achieved by enriching mRNA with a polyA tail using Oligo(dT) magnetic beads. Then, using fragmented mRNA as the template and random oligonucleotides as primers, the first strand of cDNA is synthesized in the M-MuLV reverse transcriptase system. Subsequently, the RNA strand is degraded by RNaseH, and the second strand of cDNA is synthesized using dNTPs as raw materials in the DNA polymerase I system. The double-stranded cDNA after purification undergoes end repair, addition of an A tail, and connection of sequencing adapters. The cDNA with a size of approximately 250-300 bp is selected using AMPure XP beads. PCR amplification is performed, and the PCR products are purified again using AMPure XP beads. Finally, the library is obtained. The reagent kit used for library construction is NEBNext Ultra RNA Library Prep Kit for Illumina. RNA-Sequencing (RNA-Seq) analysis was performed using illumina NovaSeq 6000 (illuminaUSA). After filtering out the reads with connectors and of low quality, the clean reads were aligned to the reference genome using STAR (version 2.7.10a). The quality control software used was fastp (version 0.19.7), with the parameters set as fastp -g -q 5 -u 50 -n 15 -l 150.