T cell receptor beta chain (TRB) sequencing of CD8 T cells from LNs

Mice were subcutaneously injected with equivalent doses of aM-exos and aMT-exos (30 μg, 3.69×10⁹ particles). 3 days later, the LNs were removed from mice and prepared to single cell suspension. Then, CD8 T cells were sorted out using CD8 Dynabeads and used for RNA isolation. RNA samples were analyzed by High-throughput sequencing of TRB using the ImmuHubTCR profiling system (ImmuQuad Biotech). Briefly, a 5’ RACE unbiased amplification protocol was used. This protocol uses unique molecular barcodes introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Sequencing was performed on an Illumina NovSeq® system with PE150 mode (Illumina). One common adaptor with UMB was added on the 5’of cDNA during the first-strand cDNA synthesize and one reverse primer corresponding to the constant (C) regions of each of the TRB were designed to facilitate PCR amplification of cDNA sequences in a less biased manner. The UMB attached to each raw sequence reads were applied to correct PCR and sequencing errors correction and PCR duplicates removing. Map V, D, J and C segments with IMGT® and then extract CDR3 regions and assemble clonotype for all clones. The resulting nucleotide and amino acid sequences of CDR3 of TRB were determined and those with out-of-frame and stop codon sequences were removed from the identified TRB repertoire. We further defined amounts of each TRB clonotype by adding numbers of TRB clones sharing the same nucleotide sequence of CDR3.