*2.6. ISE6 Cell Culture, Infection with B. burgdorferi, and Validation of Differentially Expressed miRNAs by qRT-PCR*

Differentially expressed miRNAs in RNA-seq data were validated by qRT-PCR in the ISE6 cell line derived from *I. scapularis* embryos cultured and maintained as suggested by Munderloh and Kurtti [41]. The *B. burgdorferi* (strain B31) isolate was a kind donation from Dr. Monica Embers (Tulane University, Covington, LA, USA). Cultures were grown and maintained as suggested previously [34]. Once ISE6 cells reached confluency, they were infected with *B. burgdorferi* as described previously [42]. Briefly, ISE6 cells were inoculated with the supernatant of a log phase *B. burgdorferi* culture at a multiplicity of infection of 50 and incubated for 24 h before harvesting. RNA was isolated using the TRIzol method and the expression of miRNAs analyzed by qRT-PCR with the Mir-X miRNA qRT-PCR TB Green kit (Takara Bio Inc, Kusatsu, Shiga, Japan; catalog #638316). qRT-PCR conditions were an initial denaturation at 95 ◦C for 10 min then 40 cycles of 95 ◦C for 5 s, 60 ◦C for 20 s.

## *2.7. Normalization, Differential Expression (DE), and Statistical Analysis of miRNAs between Uninfected and Infected Salivary Glands*

Differential expression (DE) analysis of identified miRNAs was performed using the interactive web interface DeApp [43]. Low-expression genetic features were removed after alignment if the counts per million (CPM) value was ≤1 in less than two samples. Sample normalization and a multidimensional scaling (MDS) plot are shown in Supplementary Figure S2. DE analysis was performed with edgeR with a false discovery rate (FDR) adjusted *p*-value of 0.05 and minimum fold-change of 1.5. DeApp displays a dispersion plot showing the overall DE analysis along with statistical significance (*p*-value, FDR adjusted *p*-value) and a volcano plot corresponding to the specified parameters and cutoff values.

## *2.8. In Silico Mapping of B. burgdorferi-Infected Small RNA Sequences to the Borrelia burgdorferi Genome*

In silico mapping of small RNA sequences of *B. burgdorferi*-infected salivary glands to the *Borrelia burgdorferi* genome (GCF\_000181575.2\_ASM18157v2\_genomic.fna) detected 18,165 *Borrelia burgdorferi* sequences, but no miRNAs of *B. burgdorferi* origin were predicted by miRDeep2 analysis.
