title: RNA-seq of homo sapiens: tumor and non-tumor renal parenchyma
design: Quality and integrity check - Qubit 2.0 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), Agilent 2200 TapeStation system and RNA ScreenTape (Agilent, Santa Clara, CA, USA). DNAse treatment - DNA-free DNA Removal Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.  Ribosomal RNA depletion - 26 ul of nuclease-free water (Qiagen, Hilden, Germany) to achieve 500 ng of total RNA input, RiboCop rRNA Depletion Kit V1.2 (Lexogen, Vienna, Austria) according to the manufacturer's protocol . Library preparation using Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), AM-Pure XP Beads (Beckman Coulter, Brea, CA, USA), and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (New England Biolabs, Ipswich, MA, USA). Minor adjustments have been made to the manufacturer's protocol. All 96 samples have been divided into eight groups, so twelve patients have been sequenced in each run. Also, we stratified samples semi-randomly, as to have tumor and non-tumor tissue and early as well as late relapse patients in each run to minimize batch effect. Samples have been pooled equimolar at the 4 nM concentration, which has been rechecked using the Qubit 2.0 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) fluorometer. The size of each pool was checked using Agilent 2200 TapeStation system and High Sensitivity D1000 ScreenTape (Agilent, Santa Clara, CA, USA) according to the manufacturer's protocol. Pools containing fragments of undesirable length have been purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Following the Illumina Denature and Dilute protocol, the polls have been denatured and diluted to 1,8 pM final concentration and loaded onto a sequencing cassette from NextSeq 500/550 High Output v2 kit, 75 cycles (Illumina, San Diego, CA, USA) and run according to the manufacturer's protocol.