Treg TCRa sequencing of mus musculus: young female thymus
All isolated total RNA (50-500 ng) were used for cDNA synthesis using TCRa and TCRb constant region specific primers ac1R and bc1R, SMARTScribe reverse transcriptase (CloneTech), and SmartN oligos for template switching at the 5 end in order to incorporate a unique molecular identifier (UMI) and MISS sequence for PCR (Table S1). The cDNA products were treated with uracil-DNA glycosylase (NEN biolab) at 37oC for 30 min to remove SmartN oligos. Three rounds of PCR using high fidelity Platinum Taq DNA Polymerase (ThermoFisher Scientific) were performed to prepare libraries for sequencing. The first PCR (18 cycles) was to enrich TCRs using MISS and TCR constant region primers (ac2R and bc2R) and primers were removed by the QIAquick PCR purification kit (Qiagen). The second PCR (20 cycles) was to add an 8 bp sample barcode to each sample at 5 end and internal constant region primers. PCR products were separated by 2% agarose (UltraPure, ThermoFisher Scientific) gel electrophoresis and DNA fragments (400800 bp) were further purified by a QIAquick Gel Extraction kit (Qiagen). Purified DNAs of each sample were quantitated by a BioAnalyser (Agilent) and combined for the third round of PCR which incorporates the Illumina adaptor. Amplified DNA was separated by 2% agarose gel electrophoresis and further purified by QIAquick Gel Extraction and followed by the PCR purification kits. The amount of purified DNA was measured using a Qubit flunrometer (ThermoFisher). Fifty picomoles DNA were used for sequencing on an Illumina HiSeq 2500 system. A modified paired end sequencing protocol was used: TCR-specific sequencing primers TRA and TRB were used for first round sequencing of 150 bps. Illumina RD2 primer was used for second round sequencing of 50 bps covering the sample barcode and UMI.