CD4+YFP+ cells from heterozygous female Bcl11b f/fFoxp3YFP-Cre/+ (het-KO) or littermate Bcl11bf/+ or +/+Foxp3YFP-Cre/wt (het-WT) mice were sorted at >97% purity using a BD Moflo-XDP cell sorter. Total RNA was extracted using the RNeasy Micro Kit (Qiagen-74004). Integrity and concentration of purified RNA were checked using the Agilent RNA 6000 Nano Kit (5067-1511) in Agilent BioAnalyzer and Nanodrop, respectively. Library was prepared from total RNA using the TruSeq Stranded Total RNA kit (Illumina-RS-122-2001) following the manufacturer’s protocols. Briefly, cDNA was made from total RNA followed by adenylation, barcoded adaptor ligation, and polymerase chain reaction (PCR) amplification (15 cycles). Libraries were size-selected for 200- to 300-bp fragments using Agencourt AMPure XP beads (A63880), and size distribution was confirmed using the Agilent High Sensitivity DNA Kit (5067-4626) in Agilent BioAnalyzer. Equivalent amounts of barcoded libraries were pooled and sequenced using HiSeq 2500 or MiSeq (Illumina) instruments.

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