title: GSM4673450: I5, E. coli, in vivo (SSII); Escherichia coli; OTHER
library_construction_protocol: Cell pellets were resuspended in 100 µl of Buffer A [10 mM Tris pH 8.0; 100 mM NaCl], supplemented with 5 U SUPERase•In™ RNase Inhibitor, by vigorously vortexing for 30 sec. 25 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0], supplemented with 100 µg/mL final lysozyme, were then added, followed by 30 sec vortexing. Samples were incubated 1 min at room temperature, after which 125 µl of Buffer C [0.5% Tween-20; 0.4% Sodium deoxycholate; 2 M NaCl; 10 mM EDTA] were added to lyse protoplasts. Samples were then incubated at room temperature for 5 min, after which 100 µl of lysate were transferred to 1 mL ice-cold QIAzol and thoroughly mixed by vortexing. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min. Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 µl (for SSII and HIV RT) or 2.5 µl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 µl 20 µM random hexamers and either 0.25 µl (for SSII) or 0.5 µl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 µl. For HIV RT, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 40 mM MgCl2], 0.5 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT (Worthington Biochemical Corporation, cat. HIVRT). For TGIRT-III, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 0.25 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT (InGex, cat. TGIRT-50). For SSII, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 µl DTT 0.1 M, 0.25 µl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III), or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 µg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, cat. P9599) in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), following manufacturer instructions.
attributes.tag: GEO Accession
attributes.value: GSM4673450