title: GSM6703513: Sorted Naive cells AF_TN1; Homo sapiens; RNA-Seq
library_construction_protocol: Mouse Tregs, exTregs, Tcons, human Tregs, Tnaive and Th1 were directly sorted in 750uL of TRIzol LS (ThermoFisher). Next, RNA was extracted using the miRNeasy micro kit (Qiagen). RNA quality was measured by using 2100 TapeStation (Agilent  Technologies). Samples with high quality RNA (RNA integrity number &gt;8.0) were used for the  next steps. Low-input human exTregs were sorted in duplicate (1,000 cells twice) in 20uL of  lysis buffer as described previously 25 . For both TRIzol LS and low-input samples, double-  stranded (ds)-cDNA was prepared using the SuperScript II reverse transcriptase  (ThermoFisher) according to the manufacturer's instructions. cDNA was amplified using 15  cycles and eluted in 24 μl. 100ng of resulting cDNA was processed using the Illumina DNA Prep  Kit (Cat#20018704, Illumina) following manufacturer's instructions. Samples were pooled and  loaded onto an Illumina NovaSeq6000, and sequenced with 50 base pair paired-end reads  (PR50).