RNA-Seq of non-responder 4 PBMCs-after treatment
RNA extracted from PBMCs and renal tissue was quantified using Qubit RNA Broad Range or High Sensitivity assay kits, purity was checked and its integrity was assessed. Ribosomal RNA was removed from 100ng of total RNA using biotinylated, target-specific oligos combined with Ribo-Cop rRNA removal beads. The RNA fragments were copied into first strand cDNA using reverse transcriptase and second strand synthesis was performed using DNA Polymerase I and RNase H enzyme. cDNA was then ligated with adapters, amplified further and checked for purity. Libraries prepared using the NEB Ultra II directional RNA-Seq Library preparation kit were pooled and loaded on Illumina Novaseq 6000 platform to generate 150bp paired end reads at a depth of forty million reads.