Taqman assays

sc-qPCR was performed on preamplified cDNA (Illumina Nextera XT dual index library prep kit following the Smart-Seq2 protocol) for 21 genes and 112 cells. Applied Biosystems TaqMan assays (FAM-MGB) were used and reactions were run on a Fluidigm Biomark microfluidic qPCR chip (PN 100-6170 C1). Spearman’s ranked coefficient was used for estimating covariations between sc-qPCR and single-cell RNA sequencing data. Limit of detection (LoD) was set at 39 cycles for covariations. Importantly, the results were overall consistent when using lower LoDs (incl. 25 cycles).

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was performed on RNA obtained from harvested ear skin. Complementary DNA (cDNA) was generated using the Superscript III Reverse Transcriptase First Strand Synthesis System (Invitrogen) using the manufacturer’s protocol with random hexamers. Samples were pre-amplified using pooled Taqman assays (ABI) and PreAmp Master Mix (Fluidigm) according to the Gene Expression Preamplification with Fluidigm PreAmp Master Mix and Taqman assays protocol (Fluidigm). Expression of 48 control and inflammatory genes was assessed using a 48x48 qPCR dynamic array (Fluidigm), with a panel of Taqman assays (ABI), according to manufacturer’s instructions. Data were calculated by the ΔΔC_T method, using either GAPDH or GUSB in each experiment to normalize transcripts within samples. Each normalized ΔC_T value was compared to the average ΔC_T for the same transcript in sham injected mice to get the -ΔΔC_T, yielding the log₂(fold change).

Single-cell gene-expression experiments were performed using 96.96 Dynamic Array (Fluidigm Inc.). Single cells were sorted into individual wells of 96-well PCR plates and preloaded with 5 µl/well of CellsDirect One-Step qRT-PCR mix (Thermo Fisher); then each well was supplemented with 1 µl of SuperScript-III RT/Platinum Taq (Invitrogen) and a mixture of 96 pooled TaqMan assays (Fluidigm Inc., 5 µM each). Single-cell mRNA was reverse transcribed (50°C for 15 min, 95°C for 2 min) and pre-amplified for 22 cycles (95°C for 15 s, 60°C for 4 min). Libraries and probes were transferred into 96.96 Dynamic Array integrated fluidic circuits (IFCs) for qRT-PCR on a BioMark HD instrument (both Fluidigm Inc.) following the manufacturer’s instructions.