Transciptor first strand cdna synthesis kit

The RNAprep Pure Cell/Bacteria Kit (TIANGEN Biotech, Beijing, China) was used for total RNA extraction according to the manufacturer’s specifications. The concentrations of RNA were quantified using an Eppendorf BioPhotometer Plus (Eppendorf, Hamburg, Germany). Reverse-transcription reactions were performed using a Transciptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s protocol on a T100^TM Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, United States).

The total RNA extraction kit (TIANGEN) was used to extract total RNA from lymphocytes in strict accordance with the operation steps in the instructions. We obtained mononuclear cells from 2ml peripheral blood using the TRIzol reagent according to the manufacturer's protocol. The concentrations of RNA were evaluated using a NanoDrop ND‐2000 spectrophotometer (Thermo Scientific). A total of 200ng RNA was converted into cDNA by reverse transcription using a Transciptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocol on a T100TM Thermal Cycler (Bio‐Rad Inc).²⁴ Quality inspection was carried out with Agilent2200, and cDNA was stored at −80°C.
Then, the libraries were constructed following the protocol from Liang et al.²⁵ For TCRβ CDR3 library preparation, two‐round nested amplicon arm‐PCR was performed with a Multiplex PCR Assay Kit Ver.2 (TaKaRa) using specific primers against each variable and constant gene. PCR products were purified by agarose gel electrophoresis, amplified using Illumina sequencing primers with different sample barcodes and subjected to high‐throughput sequencing using the Illumina HiSeq X Ten PE150 platform.