GSM3463229: DMS-MaPseq on ex vivo native deproteinized E. coli total RNA (Replicate #1); Escherichia coli; OTHER
Cell pellets from exponential phase E. coli were resuspended in 1 ml of Resuspension Buffer [15 mM Tris-HCl pH 8.0; 450 mM Sucrose; 8 mM EDTA], and 18,500 U of Ready-Lyse Lysozyme (Epicentre, #R1810M) were added. After incubation at 22 °C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 x g (4 °C) for 5 min. Pellets were resuspended in 120 µl Protoplast Lysis Buffer [50 mM HEPES pH 8.0; 200 mM NaCl; 5 mM MgCl2; 1.5% SDS]. Samples were incubated for 5 min at 22 °C and for 5 min on ice, followed by addition of 30µl SDS precipitation buffer (50mM HEPES pH8, 1M KOAc, 5mM MgCl2). The precipitate was removed by centrifugation at 17000g (4 °C) for 5min and the supernatant was subjected to gel filtration on ProbeQuant G-50 Micro Columns (GE Healthcare, #28-9034-08) pre-equilibrated 3 times with RNA folding buffer (RFB) [50 mM HEPES pH 8.0; 200 mM KOAc; 5 mM MgCl2]. The eluate was then extracted 2 times with phenol:chloroform:isoamyl alcohol (25:24:1, pre-equilibrated 3 times with RFB), and once with chloroform. 20 U of SUPERase In RNase Inhibitor (ThermoFisher Scientific, #AM2696) were added. 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.