design: For extraction of total RNA, 20-30 mg of feline tissue was added to 600 ul buffer RLT (Qiagen) with 10 ul beta-mercaptoethanol in Lysing Matrix D tubes (MP Biomedicals) and homogenised using a FastPrep-24 Classic bead beating grinder and lysis system (MP Biomedicals). RNA was purified from the resulting homogenate using a RNeasy Plus Mini kit (Qiagen), including column-based elimination of genomic DNA. Due to low A260/230 ratios in some cases, all samples were then cleaned up using an RNA Clean & Concentrator-25 kit (Zymo). Feline TCR libraries were prepared from total RNA as in PMID:31906850, with modifications. For each RNA sample, 1 ug total RNA was converted to cDNA by 5 RACE using SMARTScribe Reverse Transcriptase (Takara Bio). Each reaction included 0.5 uM anchored oligo(dT) VN and 1 uM template switch oligonucleotide (TSO). Resulting cDNA was cleaned up using AMPure XP beads (Beckman). Each TCR locus (TRA, TRB, TRG and TRD) was amplified in a separate PCR reaction using KAPA HiFi HotStart ReadyMix (Roche) with the following conditions: 95C for 3 min, 25 cycles of 98C for 20 s, 64C for 15 s, 72C for 30 s, and a final extension step at 72C for 3 min. First round PCR products were pooled and purified using AMPure XP beads at 0.8X sample volume and eluted in 50 ul ddH2O. 5 ul of the purified product was used in a second round of PCR to incorporate adapter sequences and combinatorial dual indexes according to the instructions available at https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf. Libraries were purified using AMPure XP beads at 0.5X sample volume, and library concentrations were determined using a NEBNext Library Quant Kit for Illumina (NEB). Libraries were pooled in equimolar amounts and gel extracted using a QIAquick Gel Extraction Kit (Qiagen) to select fragments at the expected size of ~600 bp, followed by a final clean up with a MinElute PCR Purification Kit (Qiagen). Feline TCR libraries were sequenced as 300 bp paired-end reads on a MiSeq instrument (Illumina).