title: GSM6659086: 48_CD25- B4-2,MHCII-Block,IL2 (3x); Mus musculus; RNA-Seq
library_construction_protocol: Treg and Tcon cells were purified from total splenocytes using a commercial mouse Treg isolation kit (Miltenyi # 130-091-041) following the manufacturer's protocol. Individual mice were processed as separate samples. Tcons were collected as the flow-through fraction in the CD25 positive selection step. The bound fraction was eluted and collected as the Treg cells. Sorted CD25 or Treg cell RNA was extracted by using Qiagen RNeasy mini kit (Qiagen #74106). The concentration and quality of the isolated RNA samples were assessed and determined on ThermoFisher NanoDrop 8000 spectrophotometer and Agilent 4200 TapeStation system, respectively, and had RNA integrity numbers at least 9.1. 200ng or 500ng RNA was used to prepare cDNA library by using a protocol modified from the Illumina TruSeq Stranded mRNA kit (Illumina #20020595). The constructed libraries were amplified and barcoded using the PCR program: a denaturation step at 98oC for 30 seconds, 15 cycles of 95 oC for 10 seconds, 60 oC for 30 second, 72oC for 30 seconds, and a 72 oC extension cycle for 5 minutes followed by a hold step at 4 oC. The amplified products were subjected to a clean-up step using AMPure XP bean (Beckman Coulter #63881) at the 0.85x ratio of beads to the PCR reaction volumes. The final barcoded libraries were sequenced to a minimum depth of 30 million paired-end reads at the 150nt read length on an Illumina Nextseq2000.