title: GSM5260429: 4.naive.CD45+TCR+CD4+Fox+3+TSLPR-Il1rl1-; Mus musculus; RNA-Seq
library_construction_protocol: Colon tissue was cut into small pieces and treated with 2 mM EDTA, followed by digestion with collagenase D (50 μg/mL) (Sigma-Aldrich) and DNase (20 units/mL) (Sigma-Aldrich) in HEPES buffer for 30 min.  Cells were filtrated through a 100μm filter to make single cell suspensions, subjected to staining with fluorescent-labeled antibodies for flow cytometry. T cell subsets were sorted on a BD FACSAria II cell sorter (BD Biosciences).  Cell subsets (200 cells per sample) were sorted directly into SMART-Seq v4 lysis buffer (Takara Bio) to release RNA. Reverse transcription was performed followed by PCR amplification to generate full-length amplified cDNA.  Sequencing libraries were constructed using a modified protocol of the Nextera XT DNA Sample Preparation Kit (Illumina) to generate Illumina-compatible barcoded libraries Libraries were pooled and quantified using a Qubit Fluorometer (Life Technologies).  Dual-index, single-read sequencing of the pooled libraries was carried out on a HiSeq 2500 sequencer (Illumina) with 58-base reads, using HiSeq v4 Cluster and SBS Kits (Illumina) with a target depth of 5 million reads per sample.