Dnase digestion

Total RNA was extracted from flash-frozen distal ileum tissues by TRIzol extraction and further cleaned using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). DNA was removed by on-column DNase digestion (Qiagen). Purified RNA was quantified using a Nanodrop 2000 spectrophotometer.

RNA extractions were performed using a miRNeasy RNA extraction kit (Qiagen, cat. no. 217084), with an on-column DNase digestion (Qiagen, cat. no. 79254). RNA concentrations were measured on a NanoDrop 1000 spectrophotometer (Thermo Scientific). For qRT-PCR, RNA samples were diluted to a consistent concentration then cDNA was prepared in duplicate using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, cat. no. 4368814) with random primers. A control “no RT” reaction was performed in parallel to confirm that all genomic DNA had been degraded. qRT-PCR was performed on each cDNA replicate in duplicate (i.e. 4 technical replicates in total), using TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies, cat. no. 4324018) and an appropriate custom designed TaqMan pre-miRNA assay (Life Technologies). For custom pre-miRNA assays, the pre-miRNA hairpin sequence (miRBase) was submitted to Life Technologies' web-based Custom TaqMan Assay Design Tool as in⁵ (link) (Fig. S4). RpL32 (Rp49) was used for normalization. For miRNA qRT-PCR, RNA was diluted to 2 ng/μl then cDNA was prepared in duplicate from 10ng RNA using a TaqMan miRNA Reverse Transcription Kit (Life Technologies, cat. no. 4366596) with miRNA specific primers. As above a no RT reaction was performed in parallel. qRT-PCR was performed as above. snoR442 was used for normalization.

RNA from non-CNS tissues was isolated with the RNeasy Mini kit with on-column DNase digestion (Qiagen). RNA from CNS tissues was isolated with TRIzol (Ambion) and digested with DNase I (InvitroGen). Gene expression was determined by reverse transcriptase quantitative PCR (RT-qPCR), using the SYBR Green (Agilent Technologies) and TaqMan (Applied Biosystems) systems. 50 ng of RNA was used for each reaction. Expression levels were quantified relative to the expression of GAPDH, using the $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_T}$ method [80 (link)], and normalized to the control group as a fold change. Data are represented as scatter plots with the mean ± SD of biological replicates. The following TaqMan primers were used: mIfnb (Mm00439552_s1; Applied Biosystems), mMx1 (Mm00487796_m1; Applied Biosystems). The following SYBR Green primers were used: mGapdh (FW: 5’-CAA TGT GTC CGT CGT GGA-3’; RW: 5’-GAT GCC TGC TTC ACC ACC-3’), mA20 (FW: 5’-TGC AAT GAA GTG CAG GAG TC-3’; RW: 5’-TGG GCT CTG CTG TAG TCC TT-3’), mIl6 (FW: 5’-GAA AAT CTG CTC TGG TCT TCT GG; RW: 5’-TTT TCTG ACC ACA GTG AGG AAT G), mTnfa (FW 5’-CAC AGC CTT CCT CAC AGA GC; RW: 5’-GGA GGC AAC AAG GTA GAG AGG).

For sequencing purposes, RNA was extracted using Trizol LS Reagent (Thermo Fisher, Waltham, MA, USA) in combination with RNeasy Mini Kit (Qiagen, Hilden, Germany) including DNase digestion (Qiagen) on the spin column.
Formalin-fixed paraffin-embedded (FFPE) materials of the squirrel including samples of lung, brain, heart, liver, kidney, spleen, and intestine were extracted using the RNeasy FFPE Kit (Qiagen) as described by the manufacturer.
For screening purposes, nucleic acids of squirrel samples were extracted using a combination of Trizol Reagent (Thermo Fisher) and automated extraction with the NucleoMagVet kit (Macherey-Nagel, Düren, Germany). Sample materials were lung, brain, swabs, or organ pools.