GSM8112555: fCLIP Membrane N-IP_Replicate2; Chlorocebus aethiops; RIP-Seq
Vero cells were infected with SARS-CoV-2 at an MOI of 0.05 for 48 hours. Infected cells were washed with cold 1x PBS, treated with 0.1% paraformaldehyde (Pierce, 28906), and added 1.5M glycine quenching solution. After washing with 1x PBS, subcellular fractionation of the cytosol and membrane was performed as described (Thermo Fisher Scientific, 78840). Each fraction was incubated with RNase A (Thermo Fisher Scientific, EN0531) for 10 minutes for RNA fragmentation. Three percent in volume of each subcellular fraction was kept as corresponding input samples. Lysates were incubated with antibodies-conjugated sepharose beads (A/G, GE Healthcare, 17-5138-01/17-0618-02) for immunoprecipitation. Beads were washed stringently, and RNAs were liberated with Proteinase K and 7M urea at 60°C overnight according to the guidelines approved by the International Vaccine Institute (IVI). RNAs were purified followed by DNase I (Takara, 2270A) treatment, and rRNAs were removed by Ribo-Zero kit (Illumina, TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat, 20020596). RNAs were ligated to the adaptor at 3ʹ-end, resolved on 6% denaturing urea-PAGE gel, and isolated from the corresponding region (50-500nt). RNAs were ligated to the 5′ adapter followed by reverse transcription and PCR amplification. fCLIP-seq libraries were sequenced by paired-end run (151 × 151 cycles) on the Illumina platform (MiSeq) with PhiX control library v.3 (Illumina).