TCR-seq of homo sapiens: adult female gd TIL
High-throughput sequencing of the expanded TIL populations was conducted using the iR-RepSeq+ service provided by iRepertoire, Inc. Sample RNA was isolated using the RNeasyextraction kit. Two independent next-generation sequencing libraries were generated for each sample, encompassing the human TCR chains. The sequencing protocol involved the initial utilization of primer pairs for each V-J combination, enabling extension with tags for subsequent global amplification of all TCR chains. The amplification process was conducted in a single assay, incorporating unique molecular identifiers (UMIs) during the reverse transcription step to distinguish between individual RNA molecules and minimize the impact of PCR duplicates and sequencing errors. Reverse transcription was performed using the OneStep RT-PCR mix, followed by selection of first-strand cDNA and removal of remnant primers using SPRIselect bead selection. Subsequently, a second round of binding and extension was carried out with a V-gene primer mix, and SPRIselect beads were employed to purify the first and second-strand synthesis products. Library amplification was achieved using primers specific to communal sites engineered onto the 5 end of the C- and V-primers employed in the initial synthesis steps. The final constructed library included Illumina dual index sequencing adapters, a 10-nucleotide UMI, and an 8-nucleotide internal barcode associated with the C gene primer. Amplified libraries were multiplexed and pooled for sequencing on an Illumina NextSeq 1000 P2 300-cycle using single-end reads. The resulting T cell receptor sequencing (TCR-Seq) data covers the second framework region through the beginning of the constant region, including the CDR3 hypervariable region.
SUB15338424
508gdr