title: GSM4673446: NIC, E. coli, ex vivo (SSII); Escherichia coli; OTHER
library_construction_protocol: Samples were then vortexed briefly and 1 mL of ice-cold QIAzol Lysis Reagent was added to each sample, followed by extensive 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: GSM4673446