GSM3463236: DMS-MaPseq on in vivo-purified Influenza A vmRNA (Replicate #2); Influenza A virus; OTHER
50 mg of total RNA were used for poly(A)-enrichment with Dynabeads Oligo(dT)25 (Thermo Fisher Scientific, #61002). The isolated poly(A) RNA was used as input for the viral mRNA capture using biotinylated DNA probes (1 probe for each segment with a length of 80 nt complementary to viral mRNA, for sequences refer to Table S1). 100 ml of Dynabeads MyOne Streptavidin T1 beads (ThermoFisher Scientific, #65601) were washed twice with 500 µl Wash Buffer 2 [100 mM NaOH; 50 mM NaCl], twice with 500 µl Wash Buffer 3 [100 mM NaCl], and resuspended in 200 ml 2X Binding & Wash Buffer 1 [10 mM Tris-HCl pH 7.5; 2 M NaCl; 1 mM EDTA]. 5 ml of an equimolar mix of all 8 probes (100 mM pool) in 200 ml water were denatured at 85 °C for 3 min, then immediately placed on ice and added to the washed beads. Probes were immobilized on beads for 20 min at 37 °C, with vigorous shaking. Beads were then washed twice with 500 µl 1x Binding & Wash Buffer 1 and resuspended in 100 µl RNA Binding Buffer [20 mM Tris-HCl pH 7.5; 1 M LiCl; 2 mM EDTA]. To the poly(A)-enriched RNA in 100 µl water, 100 µl RNA binding buffer were added and the sample was denatured at 70 °C for 5 min and immediately placed on ice. Washed beads, supplemented with 40 U of SUPERase In RNase Inhibitor, were then added to the poly(A) RNA and binding was allowed to occur at 37 °C for 2 h, with vigorous shaking. Samples were washed twice with 1 ml of Wash Buffer A (10 mM Tris-HCl pH 7.5; 150 mM LiCl; 1 mM EDTA; 0.1 % SDS] and once with 1 ml Wash Buffer B [10 mM Tris-HCl pH 7.5; 150 mM LiCl; 1 mM EDTA]. Beads were resuspended in 100 µl 95% formamide and 10 mM EDTA pH 8.0, and incubated at 90 °C for 10 min. Eluted RNA was separated from the beads and purified with 1 ml of TRIzol. To remove residual DNA probes, the eluate was treated with DNase I for 15 min at 37 °C and further purified on RNA Clean & Concentrator-5 columns. 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.