title: GSM9269303: Donor 1237, stimulated with EBNA; Homo sapiens; OTHER
library_construction_protocol: RNA was extracted from pellets of expanded CD4 cells and circulating CD4 cells using the RNeasy PLUS microkit (Qiagen) RNA was reverse transcribed using a TCRB chain constant region reverse primer with Superscript IV First-Strand Synthesis System. cDNA was column purified with the Oligo Clean Concentrator kit. cDNA of TCRB chain was used as template for PCR with KAPA2G Fast Multiplex Mix. Forward TCRB primers were used with a single nested TCRB constant region reverse primer. Samples underwent 20 cycles for NGS library preparation for PCR1. For PCR2, 2 µL of PCR1 was added to 18 µL of PCR2 master mix (0.25 µM each i5 and i7 sample barcoding primers) and underwent 20 cycles with the use of Herculase II fusion DNA Polymerase. PCR2 product was then run on agarose gel (2%) and desired band were purified using a GeneJet PCR Purification kit (ThermoFisher Scientific).