Fig. 1: TCR β chain enrichment strategy for use with 10x scRNA sequencing.

The TCR V(D)J enrichment strategy is depicted using the β chain for illustration (see Supplementary Fig. 1 for α chain). At top, the genomic un-rearranged TRB locus is shown (Note: TRB is located on the negative strand in dogs). During T cell development from progenitor T cells to mature T cells, individual V, D, J and C gene segments are rearranged by somatic recombination to produce a functional TRB locus. Transcription and splicing produce a pre-mRNA and then mRNA for the complete V(D)JC transcript sequence (not shown). In the modified 10x protocol, mRNA (including TRB mRNA) is converted to cDNA. TRB cDNA is then amplified using a nested PCR design. The forward primers from the 10x protocol were left unchanged (v2 protocol shown). In the first cycle, the forward primer (TRB Forward 1) primes off the Illumina read 1 (R1) sequencing adapter that is incorporated during generation of cDNA. In the second cycle, the identical forward primer (TRB Forward 2) again primes off the R1 sequence. The first reverse primer (TRB Reverse 1, Outer) primes off the constant (C) region gene segment. The second reverse primer (TRB Reverse 2, Inner) similarly primes off the C region but at an inner, 5’ position relative to the outer primer. The β chain primer design was based off a dog TCR β rearranged partial mRNA (GenBank: HE653957.1) which was extended to include (from 3’ to 5’) the R1 adapter, 10x cell barcode, UMI, TSO, V, D, J, and C gene segments. The constructed cDNA sequence was then used as input to primer3plus (4.0), with forward primers provided as described above, and a target region for reverse primer specified in the C region. The product of the first (outer) design was used as input for the second (inner) design.