Illumina HiSeq 2500 sequencing; RNA-seq of Zbtb20-expressing Tregs against activated (CD62Llo) and naive (CD62Lhi) Tregs that do not express Zbtb20
Spleen cells from four Zbtb20-GFP; FoxP3-RFP double reporter mice were FACS sorted using the expression of GFP, RFP, CD62L, and CD4 to obtain highly pure (~99%) populations of Zbtb20-expressing FoxP3+ Tregs, Zbtb20-negative FoxP3+ CD62L-low Tregs, Zbtb20-negative FoxP3+ CD62L-high Tregs. Approximately 100K cells were resuspended in 100uL of TRIzol  (Sigma-Aldrich) solution and stored at -80oC until the RNA extraction. RNA was isolated from the cell using the Direct-zol RNA MicroPrep Kit (Zymo Research). RNA quality, SMARTer mRNA Amplification, libraries preparation, and RNA-Seq were done at the Lewis-Sigler Institute for Integrative Genomics, Princeton University.  500 ng of cDNA was PCR amplified with Clontech SMARTerâ„¢ Ultra Low RNA Kit. Library was preparated with Illumina TruSeq according to the standard protocol.