GSM8332592: sfRNA2 , DMS treated, Rep2; Powassan virus; OTHER
Modified RNAs were cleaned up with the RNEasy Micro Kit Reverse transcription, second strand synthesis, and library prep of modified RNA. The following protocol was applied to the full length 3′UTR, sfRNA1 and sfRNA2 RNA species. Approximately 400 ng of modified or control RNA was incubated with of 200 ng/μL random nonamer primer (NEB; S1254S) and 1.0 μL of POWV_SHAPEMAP_RT_Primer (Supplemental Table S2) at 65°C for 5 minutes and then cooled on ice. To each of the reactions 8.0 μL of MAP buffer (125mM Tris pH 8.0, 187.5mM KCl, 15mM MnCl2, 25mM DTT, 1.25mM equimolar dNTP mix, and 200U Superscript III) was added to the reaction and incubated at 25°C for 2 minutes. The reaction was then incubated at 25°C for 10 minutes, 42°C for 3 hours, 70°C for 15 minutes, and cooled to 4°C. The RNA/cDNA hybrids were then purified on the G25 (Cyteva) spin column following the manufacturers protocol. Second strand synthesis of the cDNA was carried out using the NEBNext second strand synthesis kit (NEB) following the manufacturer's instructions. The resultant cDNA was purified using the DNA Clean & Concentrator-5 kit (Zymo) eluting in 10 μL of DEPC-treated H2O and quantified fluorometrically with the Qubit (Thermo) using the dsDNA HS Assay Kit (Thermo). Next generation sequencing library prep was performed with the Nextera XT DNA library preparation kit (Illumina) using the Nextera XT Index Kit v2 Set B (Illumina). Samples were normalized and pooled together using a combination of the Qubit (Thermo) and the 4200 TapeStation System (Agilent Technologies) with the High Sensitivity D1000 Screen Tape (Agilent Technologies). Final libraries were sequenced on the NovaSeq 6000 platform (Illumina).