title: GSM8335533: DMS-MaPseq of E. coli DH5a, in vitro refolded, 37°C (mRNA); Escherichia coli; OTHER
library_construction_protocol: For in cell treatments, cell pellets were resuspended in 62.5 μl Resuspension Buffer [20 mM Tris-HCl pH 8.0; 80 mM NaCl; 10 mM EDTA pH 8.0], supplemented with 100 μg/mL final Lysozyme (cat. L6876, Merck) and 20 U SUPERase•In™ RNase Inhibitor (cat. A2696, ThermoFisher Scientific), by vigorous vortexing. Samples were incubated at room temperature for 1 min, followed by addition of 62.5 μl Lysis Buffer [0.5% Tween-20; 0.4% Sodium deoxycholate; 2 M NaCl; 10 mM EDTA]. Samples were then inverted 5-10 times and incubated at room temperature for 2 min, followed by additional 2 min on ice. 1 mL ice-cold TRIzol™ Reagent (cat. 15596018, ThermoFisher Scientific) was then added, samples were vigorously vortexed for 15 sec, and RNA was extracted as per manufacturer instructions. Prior to library preparation, highly abundant short RNA species, such as tRNAs, were depleted on Monarch® RNA Cleanup columns by loading a 1:1:1 mixture of total RNA in nuclease-free water, RNA binding buffer and 100% ethanol. rRNA depletion was performed on 1.1 μg total RNA using the RiboCop for Bacteria kit (cat. 126, Lexogen), with two minor changes to the manufacturer's protocol: denaturation temperature was increased to 95°C, and probe annealing temperature was lowered to 55°C. Following rRNA depletion, RNA was cleaned up on Monarch® RNA Cleanup columns, eluted in 8 μl nuclease-free water, and supplemented with 2 μl 100 μM random hexamers, 2 μl dNTPs (10 mM each) and 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2]. Samples were then incubated at 94°C for 5.5 min, to simultaneously denature and fragment the RNA to a median size of 200 nt, and immediately transferred to ice for 1 min. Samples were then supplemented with 1 μl DTT 0.1 M, 20 U SUPERase•In™ RNase Inhibitor, 200 U TGIRT™-III Enzyme (InGex, cat. TGIRT50) and 25 ng/μl Actinomycin D (to increase strand-specificity; cat. A1410, Merck), and then incubated at 25°C for 10 min, 57°C for 1 h, and 60°C for 1 h. TGIRT-III was degraded by adding 2 μg Proteinase K and incubating at 37°C for 20 min. Proteinase K was inactivated by addition of Protease Inhibitor Cocktail (cat. P8340, Merck). cDNA-RNA hybrids were then converted to dsDNA using the NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module (cat. E7550, New England Biolabs), by incubating at 16°C for 1 h. DsDNA was then cleaned up with 1.8 volumes NucleoMag NGS Clean-up and Size Select beads (cat. 744970, Macherey Nagel), and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (cat. E7645S, New England Biolabs), as per manufacturer instructions.