GSM8022514: Donor_1195_OVA; Homo sapiens; RNA-Seq
RNA was extracted from antigen-specific T cells using the RNeasy Plus Mini Kit (Qiagen) and sample RNA was reverse transcribed using a TCRB chain constant region reverse primer with Superscript IV First-Strand Synthesis System (Invitrogen). cDNA was column purified with the Oligo Clean and Concentrator Kit (Zymo Research). cDNA was used as a template for PCR with KAPA2G Fast Multiplex Mix (Roche). Forward TCRB FR3 primers were used with a single nested TCRB constant region reverse primer. 20 cycles for NGS library preparation for the PCR1 and 20 cycles of PCR2 were performed on the PCR1 product to incorporate sample barcodes and Illumina sequencing adaptors using Herculase II Fusion DNA Polymerase (Agilent). Specific principles and methodology have been previously reported. PCR2 product underwent a run of agarose gel electrophoresis. Bands at 300pb were purified using a GeneJET purification kit (Thermo Fisher) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). RNA-seq on an Illumina NextSeq 500 with 15% of PhiX control v3 (Illumina)