title: GSM3463233: DMS-MaPseq on in vitro-transcribed denatured Influenza A vmRNA (Replicate #1); Influenza A virus; OTHER
library_construction_protocol: A T7 promoter was introduced 5' to each segment via PCR using pDZ-PB2, PB1, PA, HA, NP, NA, M1/2, NS1/NEP plasmids as a template. Segments were then in vitro transcribed using T7-FlashScribe kit (CellScript, #C-ASF3507). Transcription products were verified by capillary electrophoresis on a Fragment Analyzer (Advanced Analytical). For in vitro refolding, segments were pooled equimolarly and 240 ng of the pool were used per replicate. RNA was fragmented in 64 mM Tris-HCl pH 8.3, 96 mM KCl, 3.9 mM MgCl2 for 8 min at 94 °C and subsequently purified on RNA Clean & Concentrator-5 columns. Reverse transcription (RT) was performed using the TGIRT-III enzyme (InGex, #TGIRT50) as described previously (Zubradt et al., 2016). Briefly, to the 5 µl of fragmented RNA, 1 µl 10 mM dNTPs and 0.5 µl 10 µM random hexamers were added, and the mixture was heated to 70 °C for 5 min and immediately placed on ice for 1 min. 2 µl of 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], DTT to 5 mM, 10 U SUPERase In RNase Inhibitor, and 100 U TGIRT-III were added. Reverse transcription was then allowed to proceed at 25 °C for 5 min, followed by 1.5 h at 57 °C. To remove TGIRT-III from the RNA-DNA duplex, 1 µl of proteinase K was added to a final concentration of 0.1 µg/µl and the reaction incubated at 37 °C for 15 min., 1 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, #P8340) was then added to inhibit proteinase K activity. The reaction volume was then adjusted to 25 µl by adding 3 µl of 5X RT Buffer and 10 µl water. Libraries were then prepared using the TruSeq RNA Library Prep Kit v2 (Illumina, #15025063), starting from second strand synthesis.