Rna extraction kit

Total RNA from cell samples in each well was separately extracted using the RNA Extraction Kit (Biotek, RP4002). Purity and concentration of the RNA samples were measured using NanoDrop (Thermo Fisher). Total RNA was used to synthesize cDNA with HiScript^® 1st Strand cDNA Synthesis Kit (Vazyme, R211-02) according to the manufacture’s protocol.
For qPCR, the HiScript One Step qRT-PCR Probe Kit (Vazyme, Q222-01) was used to detect the viral RNA copies, and SYBR Green Kit was used to analyze expression of cellular genes on a Mx3005P Real-Time PCR system (Agilent, United States). Gene-specific primers are listed in Table 1. The cycling parameters included initial denaturation at 95°C for 30 s, followed by 45 cycles (5 s at 95°C and 40 s at 60°C), with a final cooling at 42°C for 30 s. The viral RNA copies were calculated as described (Zhou et al., 2019 (link)). The qPCR parameters for quantification of relative expression of cellular genes were: 95°C for 5 min for initial denaturation; 40 cycles of 95°C for 10 s, 60°C for 30 s, followed by a dissociation curve segment (95°C, 1 min; 60°C, 30 s; 95°C, 30 s). The relative expression of the host cell genes was normalized to GAPDH.

Employing the RNA Extraction Kit (Bio-Tek, Winooski, VT, United States) to extract total RNA, and mixed it with the reaction solution conFig.d with reverse transcription reagent (RT Master Mix for qPCR Kit, MedChemExpress, Monmouth Junction, NJ, United States), then put it into gradient PCR instrument for reverse transcription reaction. A total of 3 U/mg RNase R (Epicentre, Madison, WI, United States) was used to perform the RNase R treatment for 15 min at 37°C. The cDNA formed by reverse transcription was then conFig.d with SYBR green (SYBR^® Green qPCR Master Mix, MedChemExpress, Monmouth Junction, NJ, United States). The primers for mmu-miR-1247-5p, mmu-miR-214-3p and mmu-miR-199a-5p were purchased from RiboBio (China), The primers sequences for the detection of DEmRNAs (Krt80, Hba-a1, and Htr2a), DElncRNAs (lncRmst, lncC730002L08Rik, and lncGm10635), DEmiRNAs (mmu-miR-1247-5p, mmu-miR-214-3p and mmu-miR-199a-5p), and DEcircRNAs (mmu_circ_0014855, mmu_circ_0001104, and mmu_circ_0000494) and the GAPDH were shown in Table 1.

RNA Extraction Kit (Bio-Tek, Winooski, VT, USA) was used to extract total RNA, which was mixed with the reaction solution conFig.d with reverse transcription reagent (RT Master Mix for qPCR Kit, MedChemExpress, Monmouth Junction, NJ, USA), and placed into gradient PCR instrument for reverse transcription reaction. RNase R treatment was performed at 37 °C for 15 min using 3 U/mg RNase R (Epicentre, Madison, WI, USA). The cDNA formed by reverse transcription was then conFig.d with SYBR green (SYBR^® Green qPCR Master Mix, MedChemExpress, Monmouth Junction, NJ, USA). The primers for mmu-miR-370-3p were purchased from RiboBio (China), and the sequence of mmu-miR-370-3p was 5ʹ-GCCUGCUGGGGUGGAACCUGGU-3ʹ. The sequences of the primers for the detection of circIgfbp2, Igfbp2, BACH1, and GAPDH are shown in Supplementary Table 4.