Magnetic beads

Bloodstream-stage trypanosomes were grown to 10⁶ cells/mL and insect-stage parasites to 10⁷ cells/mL. In each case, 5 × 10⁸ cells were pelleted at 3000× g and washed in phosphate-buffered saline (PBS: 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH7.4, 137 mM NaCl, 2.7 mM KCl) supplemented with 20 mM glucose. Cells were shock-frozen in liquid N_2, and stored at −20 °C. Total RNA was isolated by guanidinium acid phenol extraction [26 (link)]. Polyadenylated RNA (poly(A)-RNA) was isolated from total RNA or crude cell lysates by 2 rounds of affinity chromatography using oligo d(T)₂₅-derivatized magnetic beads (NEB). RNA yields were quantified by ultraviolet (UV) spectrophotometry and the integrity of the isolates was electrophoretically analyzed in 2% (w/v) agarose gels. Poly(A)-enrichment was assessed using qRT-PCR, comparing the ratio of the amount of β-tubulin-specific mRNA over 18S rRNA to that of total RNA using the ΔΔCT method [27 (link)].

For each cell line 50,000 cells were plated in 35 mm dishes, 24 h later cells were treated (or not) with 4Gy IR (RS-2000, RadSource), 3 h after that cells were lysed with Trizol (Ambion). RNA was purified on affinity columns and DNAse treated (Zymo). Purified RNA (500 ng) was polyA purified using magnetic beads (NEB), fragmented and reverse transcribed using protoscript RT (NEB), second strand synthesized (NEB), and then assembled into libraries with the commercial NEBnext kit (NEB) and associated protocols, although reaction volumes were reduced by 4-fold and custom adaptors and barcodes were employed. Libraries were sequenced with single end 75 bp reads on a NextSeq.

For RNA-seq, macrophages were washed twice with cold PBS and lysed in TRIzol reagent (Thermo). RNA was isolated from the aqueous phase using RNeasy kits (QIAGEN). RNA with RIN > 8 was subjected to poly-dT pulldown using magnetic beads (NEB) before preparation for RNA-seq using RNA Ultra kits (NEB). Libraries were sequenced on a NextSeq 500 (Illumina) and reads were aligned to the mm10 transcriptome using HISAT2 (Kim et al., 2015 (link)) after adaptor trimming using cutadapt (Martin, 2011 ). Reads counts per gene for RefSeq genes were computed using featureCounts (Liao et al., 2014 (link)). Counts were normalized to reads per kilobase per million (RPKM) and processed for pairwise differential expression analysis of selected conditions using DESeq2 (Love et al., 2014 (link)) with a False Discovery Rate (FDR)-adjusted p value cutoff of 0.05. Gene Ontology analysis was performed using the PANTHER database (Thomas et al., 2003 (link)).

Total RNA was extracted from HepG2.2.15 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). m⁵C-RIP was performed as previously described [55 (link)]. Briefly, 300 μg of RNA were resuspend in immunoprecipitation (IP) buffer (150 mmol/L NaCl, 0.1% NP-40, 10 mmol/L Tris-HCl pH 7.4) and incubated with an anti-m⁵C antibody (ab10805, Abcam) or normal rabbit/mouse IgG antibody (Proteintech) overnight at 4 °C. The RNA and antibody mixture was then incubated with 35 μL of magnetic beads (New England Biolabs) for 2 h at 4 °C. Beads were washed with IP buffer six times and then incubated with 300 μL of elution buffer (5 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA pH 8.0, 0.05% SDS, 4.2 μL 20 mg/mL proteinase K) at 50 °C for 1.5 h. Eluted RNA was purified using phenol/chloroform. Immunoprecipitated RNA was used for cDNA synthesis using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme) according to the manufacturer’s protocol. The relative RNA level was measured by quantitative PCR (qPCR) using Hieff® qPCR SYBR® Green Master Mix (Yeasen Biotech Co., Shanghai, China) on a CFX Connect real-time system (Bio-Rad Laboratories, Hercules, CA, USA). The primers used for RT-qPCR were listed in Table S6. At least three samples in each qPCR analysis were prepared, and three independent experiments were performed.

RNA was extracted from EV71-infected Vero cells and concentrated viral supernatants using TRIzol reagent (Invitrogen) or transcribed by T7 polymerase. For acRIP, 300 μg total RNA, 10 μg supernatant RNA, or 10 μg in vitro-transcribed EV71 RNA was incubated with anti-ac4C antibodies (Abcam) or IgG antibodies (Proteintech, Rosemont, IL, USA) in 500 μl IP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris–HCl, pH 7.4) for 4 h at 4°C. The mixtures were incubated with 30 μl anti-rabbit antibodies conjugated with magnetic beads (NEB, Ipswich, MA, USA; cat. no. S1432S) for 2 h at 4°C and washed six times with 1 ml IP buffer. RNA was extracted using TRIzol reagent and quantified by qRT-PCR.

Although bees may pick up viruses on flowers and test positive for the presence of a virus, these are not necessarily active infections. To test for actively replicating viruses in the bumblebees, we conducted strand specific RT-PCR [38 (link)] on extracted RNA samples that tested positive for a virus. Each RNA sample was transcribed to cDNA using iScript cDNA Synthesis Kit (BioRad). To increase specificity, we used PAGE purified, biotinylated forward and reverse primers (Integrated DNA Technologies) during reverse transcription and purified the resulting cDNAs using magnetic beads coated with a monolayer of streptavidin following manufacturers protocols (New England BioLabs). We diluted each cDNA tenfold and then conducted PCRs with non-biotinylated primers in separate reactions for both for forward and reverse strands.