Trizol

Salivary tissues and midguts were preserved in TRIzol (Thermo-Fisher, Waltham, MA). Salivary glands were excised as described by Cicero and Brown [67 (link)] in pools of 150 per replicate in TRIzol LS (Thermo-Fisher, Waltham, MA). Samples were kept at −80°C (bioreps 1–3, CLas (-/+) were kept one year, while replicate 4, both CLas (-/+), was kept for two years) prior to RNA extraction. Total RNA was extracted for both midguts and salivary glands following the standard TRIzol (Thermo-Fisher, Waltham, MA) RNA extraction protocol [68 (link)] including light syringe disruption prior to adding ethanol, and DNase treatment to purify total RNA. Total RNA quality was tested using an RNA gel prior to library preparation. Details of midgut sample handling can be found in Kruse et al. [16 (link)].
Illumina (San Diego, CA) libraries for all samples were made by Polar Genomics LLC (Ithaca, NY) following the protocol of Zhong et al. [69 (link)] and included poly-A tailed mRNA enrichment. Libraries were shipped on dry ice to GENEWIZ (Azenta Life Sciences, Plainfield, NJ), where they were pooled for Illumina (San Diego, CA) paired-end (PE) 150 bp sequencing. Bacteriome, head, and salivary gland samples were sequenced separately from the previously published midgut samples [16 (link)]. Raw data have been uploaded to NCBI and are accessible to reviewers via BioProject accession No. PRJNA385527.

Total RNA was extracted from NSCLC cells using TRIzol^® (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions (26 (link)). Collected NSCLC cells were lysed by 1 ml of TRIzol (Thermo Fisher Scientific, Inc.). Following lysis, total RNA was extracted using the phenol-chloroform method (27 (link)). The purity of RNA was determined by UV A260/A280 spectrophotometry (Nanodrop ND2000; Thermo Fisher Scientific, Inc.). cDNA was then obtained by reverse transcription from 1 µg RNA using miScript II RT kit (Qiagen GmbH) and stored at −20°C. RT-qPCR was performed using SYBR Green PCR kit. The reaction system consisted of 10 µl RT-qPCR-mix, 0.5 µl forward primer (human PEDF forward, 5′-ATTCCCGATGAGATCAGCA-3′; and human GAPDH forward, 5′-AGCCACATCGCTCAGACAC-3′), 0.5 µl reverse primer (human PEDF reverse, 5′-CTTAGGGTCCGACATCATGG-3′; and human GAPDH reverse, 5′GCCCAATACGACCAAATCC-3′), 2 µl cDNA and 7 µl double distilled water (ddH₂O). The reaction protocol was as follows: Initial denaturation at 95°C for 10 min, and 40 cycles at 95°C for 1 min and 60°C for 30 sec. Analysis of relative gene expression data by RT-qPCR was 2^−ΔΔCq method (28 (link)).

H1-Hela cells were seeded in a 12-well plate with a density of 200,000 cells per well and incubated overnight. For virus attachment assay, cells were incubated with RV-B14 or RV-A16 at an MOI of 20 in cold medium on ice for 60 min, washed by PBS for three times and then the total RNA was extracted using Trizol (Thermo). For virus entry assay, cells were incubated with RV-B14 or RV-A16 at an MOI of 20 in cold medium on ice for 60 min, washed by PBS for three times, and then treated with pre-warmed medium for 40 min at 37 °C. Then cells were washed with PBS for three times and treated with 0.25% trypsin for 2 min (Thermo, Cat. No. 25200072) to remove surface-bound viral particles. The internalized viral RNA was extracted using Trizol (Thermo) and viral loads were determined using RT-qPCR and FISH.

Total RNA purification was conducted by direct-zol RNA miniprep (zymo research, Irvine, CA) or RNeasy mini kit (Hilden, Germany) from a cell line or homogenized tissue block of C57BL/6 mouse in TRIzol (Thermo Fisher Scientific). Blood total RNA from C57BL/6 mouse was extracted by NucleoSpin RNA blood column (Macherey-Nagel, Dueren, Germany), followed by addition of TRIzol (Thermo Fisher Scientific) and another round of purification by direct-zol RNA miniprep (zymo research). Total RNAs from human liver (No. 636531), kidney (No. 636529), lung (No. 636524), heart (No. 636532), and skeletal muscle (No. 636534) were purchased from Takara Bio (Shiga, Japan). For RT-PCR, total RNA was applied for reverse transcription by PrimeStar Reverse Transcriptase (Takara Bio) with random hexamers, and the resulting products were amplified with ExTaq DNA polymerase (Takara Bio) with target-specific primer sets. Primers used for RT-PCR are listed in Supplementary Table 2. Detection was conducted with ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) and analyzed by Image Lab software (6.0.1) (Bio-Rad).