title: TI_Treg of Patient 15
design: For a single gene combination TRBV12-4/TRBJ1-2 sequencing, total RNA was isolated from the T cell samples using TRIzol® Reagent(15596018 ,Life Technologies, Grand Island, NY) according to manufacturer’s instructions, and was reversely transcribed into first-strand cDNA using PrimeScript™ RT Master Mix (RR036A, TaKaRa Bio Inc., Yokkaichi, Japan). we amplified sequence of TRBV12-4/TRBJ1-2 clonotypes of different samples by PCR using adaptor-ligated barcode primers specific for TRBV12-4 (5' adaptor-barcode-CTGAAGATCCAGCCCTCAG-3') and TRBJ1-2 (5' adaptor-GTTAACCTGGTCCCCGAAC-3'). PCR was performed using 2×PhantaTM HS Master Mix(P512, Vazyme biotech Co., Ltd., Nanjing, China). The PCR amplification mixture contained 19 μl of water, 25 μl of 2×PhantaTM HS Master Mix, 2 μl of 10 μmol primer (each), 2 μl of cDNA. The parameters of the PCR were: one cycle of 95 °C for 3 min, followed by 35 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 20 s, and then one cycle of 72 °C for 5 min.PCR products were purified using 2% agarose gel and Gel Extraction Kit (D2500, Omega Bio-Tek, Doraville, GA, USA), the amplified DNA fragments were visualized by G-Red (EP2201, Bioteke Corporation, Beijing, China) staining and UV transillumination in agarose gel.