Generation of cirVDJseq libraries from 3’-barcoded cDNA v1

This protocol describes circVDJ-seq, a method for simplified and cost-efficient TCR VDJ profiling from 3’-barcoded cDNA generated in single-cell or single-nucleus nucleus RNA-seq, RNA+ATAC multi-omics, or spatial transcriptomics workflows. 3’-barcoded cDNA is modified with Gibson assembly overhangs, circularized, depleted of remaining linear DNA, and subjected to nested PCR and 10x Genomics VDJ library construction to generate sequencing-ready TCR libraries. The expected outcome is a discrete, high-quality VDJ library peak on TapeStation High Sensitivity DNA assays with sufficient yield for Illumina sequencing using custom primers. When applied to human tissues, circVDJ-seq enables robust recovery of TCRα/β VDJ sequences and clonal repertoires, allowing characterization of T cell clonality and immune microenvironments across diverse clinical samples.