GSM3463243: DMS-MaPseq on in vivo Influenza A vmRNA mutated structural motifs; Influenza A virus; OTHER
Cells were collected by centrifugation and total RNA extracted with TRIzol Reagent (ThermoFisher Scientific, #15596018). After phase separation, the aqueous phase was added to 1 ml of 100% ethanol and the RNA purified on RNA Clean & Concentrator-5 columns (Zymo Research, #R1016). RT reactions were performed using the TGIRT-III reverse transcriptase in 20 µl total reaction volume, using 50 µM of a specific primer (except for segment 5 for which an oligodT primer with an overhang was used). PCR was then performed on the resulting cDNA using specific primers amplifying a region of ~250-300 bp centered on the mutated domains. PCR products were pooled equimolarly and fragmented by sonication. Sonicated DNA was gel purified to remove residual full-length products and subjected to library preparation using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs, #E6240L) according to manufacturer's instructions, but omitting size selection.