Total RNA was isolated from Treg cells of spleen sorted directly into RNA lysis buffer (RNeasy, Qiagen). RNA quality was controlled by Bioanalyzer (Agilent Technologies) RNA 6000 Pico kit (Agilent Technologies) regarding RIN > 9. cDNA from each sample were subjected to tagmention and amplification using Illumina Nextera XT kit. The resulting libraries were pooled, double-sided size selected (0.5x followed by 0.8x ratio using Beckman Ampure XP beads) and quantified using an Agilent 4200 TapeStation System. The pool of libraries was sequenced in an Illumina NovaSeq 6000 sequencer (single-end 100 bp) with a depth of around 20 Mio reads per sample. amplicon