Mastercycler gradient instrument

In order to amplify dog TCR sequences, the nested PCR approach utilized for human and mouse protocols was adopted, with dog-specific reverse primers located in the constant region of the TCR cDNA (see Primer design; Supplementary Table 1). Briefly, 2 uL of cDNA were amplified in a Mastercycler Gradient instrument (Eppendorf) in 100 uL total reaction volume using Phusion high fidelity polymerase (Thermo Fisher). The first reaction consisted of an initial denaturation step (98 °C for 45 s) followed by 12 cycles of 98 °C for 20 s, 65 °C for 30 s, and 72 °C for 60 s. An additional extension step (72 °C for 60 s) was added at the end. The PCR product was processed using the double-sided SPRI bead purification protocol described in the 10x Genomics user manual for single cell 5’ reagent kits v2, CG000331 Rev C. The resulting product was amplified in the second reaction. Amplification conditions were identical except for the annealing temperature which was 62 °C. The amplification product was purified using SPRI beads before quality control on the Agilent Bioanalyzer high sensitivity chip and further processing for library generation.

Total RNA samples (see Section 2.4) were reverse-transcribed into cDNA using QuantiTect Reverse Transcription Kit (205311; QIAGEN) and Eppendorf Mastercycler Gradient Instrument. CDNA was stored at −20 °C before further use. Quantitative real-time PCR (qPCR) was performed (samples in duplicates) to verify RNA sequencing results using gene-specific primer and hydrolysis probes (Lepr: Rn01433205_m1, NM_012596.1; TaqMan^®; Thermo Fisher Scientific) on a CFX96™ Real-Time PCR Detection System. Efficiency and Cq-values of samples were calculated using LinRegPCR software (v2020.0). Efficiency-correction and normalization to reference genes (Actb (Rn00667869_m1, NM_031144.3), Ubc (Rn01499642_m1, BC103477.1)) were performed using qBase+ (Biogazelle).