RNA-seq Protocol for Kidney Cancer

Total RNA from three groups (Cnsi, Cnsi-IFNγ, and PD-L1si IFNγ) for both A498 and 786-O cell lines, was extracted using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. Quality of the RNA samples was checked using the Agilent Bioanalyzer RNA Nano Chip (Santa Clara, CA) to determine RNA Integrity Number (RIN) ≥ 8. RNA samples were processed following the Illumina (San Diego, CA) TruSeq Stranded messenger RNA Sample Preparation Kit performed at FDA Core Facility (36 (link)). Briefly, poly (A) tailed RNA was purified from 500 ng of total RNA, fragmented, and reverse-transcribed into cDNAs. Double strand cDNAs were adenylated at the 3’ ends and individually indexed, followed by limited-cycle (15 (link)) amplification. Paired-end sequencing (100x2 cycles) of multiplexed mRNA samples was carried out on Illumina NovaSeq 6000 (A498) and NExtSeq 500 (786-O) sequencers. Sequencing reads raw data were converted to fastq files by bcl2fastq2 program (version 2.19.0) for subsequent data analysis. Raw fastq files and processed gene counts are available from NCBI GEO (Accession #GSE199107).