Dnase 1 treatment

Nascent EU RNA-seq (neuRNA-seq) labeling and capture were done by using Click-iT Nascent RNA Capture Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Kc cells were incubated with 0.2 mM EU for 1 h and RNA was extracted with Trizol (Thermo Fisher Scientific). Next, RNA was chemically fragmented for 5 min at 70 °C with RNA Fragmentation Reagents (Thermo Fisher Scientific), followed by DNase I treatment (Roche). Then RNA was ethanol precipitated after Phenol:Chloroform (Thermo Fisher Scientific) purification. The Click-iT reaction was performed with 0.5 mM biotin azide using 5 µg of EU-RNA, and biotinylated RNA was captured with 12 μL T1 beads. The nascent EU-RNA was used to generate RNA-seq libraries with Ovation RNA-seq Systems 1–16 for Model Organisms (Nugen). Samples were sequenced on HiSeq2500 (Illumina) using 50 bp single-end sequencing at the NIDDK Genomics Core Facility.

Total RNA was harvested using the miRNeasy Mini Kit (Qiagen) followed by DNase I treatment (Roche). 1 µg of total RNA was reverse-transcribed using SuperScript Reverse Transcriptase III (Thermo Fisher) with random primers (Promega), following manufacturer’s instructions. The qPCR primers for amplification are listed in Supplementary Table 1. For quantitative RT-PCR, 2 µl of cDNA were analyzed using a CFX Connect real-time PCR machine (BioRad) with SensiFAST SYBR Lo-ROX Kit (Bioline). Cycling parameters were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. The result was normalized to RPLP0. ΔcT was calculated as the difference in the cycle threshold of the transcript of interest and RPLP0, plotted as fold change compared to the CTR untreated sample.

Extraction of total RNA from cells after induced overnight expression was done using the RNeasy Kit with optimized protocol for extraction from E. coli adapted from RNAprotect® Bacteria Reagent Handbook (Qiagen)⁶⁹ . Specifically, cell lysis was performed enzymatically using lysozyme from chicken egg white (Sigma) and proteinase K (NEB), followed by standard RNeasy protocol with on-column DNase I treatment (Roche) and final elution in RNase-free water.
RNA extraction from the purified VLP samples was performed based on the previously published protocol of extraction from Potyvirus particles^{70 (link)}. In brief, the sample was incubated in the presence of 1% SDS (w/v) at 55 °C for 5 min, followed by phenol-chloroform extraction. The extracted RNA was then precipitated by the addition of 0.5 initial sample volume of 7.5 M ammonium acetate and 2.5 volumes of cold absolute ethanol at −20 °C for 1 h, followed by 25 min centrifugation at 12,000 × g. After washing the pellet with 70% ethanol (v/v) and air-drying for 15–30 min at room temperature, the precipitated RNA was resuspended in DEPC-treated water and treated with Turbo DNase rigorous protocol (Thermo Fisher) to remove any potential DNA contaminants, followed by isolation with RNA Clean & Concentrator-5 kit (Zymo Research) and storage at −80 °C.