Total RNA was extracted from Tregs using TRIzol reagent (Invitrogen, CA, USA). After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT)   beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM Magnetic   Kit (Epicentre, Madison, WI, USA). Then the enriched mRNA was fragmented into short   fragments using fragmentation buffer and reverse transcripted into cDNA with random   primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and   buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen,   Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing   adapters. The ligation products were size selected by agarose gel electrophoresis, PCR   amplified, and sequenced using Illumina Novaseq by Gene Denovo Biotechnology Co.   (Guangzhou, China).
GEO Accession
GSM4584335