RNA from sorted DN3b cells (LinCD25+CD44CD27+) from Tcf1−/− and WT littermate thymi was isolated using the Mini RNeasy Kit (Qiagen) The integrity (scores >9.0) of the RNA was determined on the Agilent 2100 Bioanalyzer (Agilent). Total RNA enrichment for sequencing poly(A) RNAs was performed with the TruSeq mRNA Sample Preparation Kit (Illumina). One microgram of total RNA for each sample was used for poly(A) RNA selection using magnetic beads coated with poly-dT, followed by thermal fragmentation. The fragmented poly(A) RNA–enriched samples were subjected to cDNA synthesis using an Illumina TruSeq preparation kit. cDNA was synthesized by reverse transcriptase (SuperScript II) using poly-dT and random hexamer primers. The cDNA fragments were then blunt-ended through an end-repair reaction, followed by dA-tailing. Subsequently, specific double-stranded barcoded adapters were ligated, and library amplification for 15 cycles was performed. The pooled cDNA library consisted of equal concentration barcoded samples. The pooled library was sequenced in one-lane, 360–base pair (bp) single read on HiSeq2500 (Illumina). Raw RNA-seq reads are accessible on SRA (Sequence Read Archive) by accession number SRP158670.

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