RNA-seq  of RENCA tumor aPD-1 resistant
The  Qiagen Tissue Lyser together with the RNeasy Mini Kit (Qiagen Cat. No. 74104)  was used to extract RNA from tissue.  Samples were randomized before sequencing library preparation and  sequencing. Extracted RNA was of high quality (RIN 7.8  10) and 500 ng (as  quantified using Agilent Bioanalyzer) of extracted total RNA was used for  whole mRNA-sequencing library preparation. The protocol for Illumina TruSeq  stranded mRNA library preparation (Illumina Cat. No. RS-122-2101) was  followed. The yield and quality of RNA-sequencing libraries was assessed on  the Agilent Bioanalyzer. Libraries were sequenced on the Illumina HiSeq4000  system, paired-end 2 x 51 cycles.