NextSeq 500 sequencing; RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori
The H. pylori strains used in this study are derived from strain B128 H. pylori was grown on Blood Agar Base 2 (Oxoid) plates supplemented with 10% defibrinated horse blood with an antibiotic-antifungal cocktail (2.5 μg.ml-1 amphotericin B, 0.31 μg.ml-1 polymyxin B, 6.25 μg.ml-1 trimethoprim and 12.5 μg.ml-1 vancomycin). Selection of H. pylori mutants was performed using 10 μg.ml-1 apramycin or 20 μg.ml-1 kanamycin. For liquid culture, Brucella broth supplemented with 10% Fetal Bovine Serum (Eurobio) and antibiotic-antifungal cocktail was used. H. pylori was grown at 37oC under microaerophilic conditions (6% O2, 10% CO2, 84% N2) generated with an Anoxomat (MART Microbiology) atmosphere generator. RNA was extracted from 2 mL H. pylori cultures at an OD600 nm of 0.5 in Brucella medium using a Total RNA Purification kit (Norgen Biotek Corp) and the samples were stored at -80 ºC until further processing.  The QIAseq Fast Select -5S/16S/23S (QIAGEN) kit was used for ribosomal RNA depletion according to manufacturer instructions. Libraries were built using a TruSeq Stranded mRNA library Preparation Kit (Illumina, USA) following the supplier's recommendations. Quality control was performed on an Agilent Bioanalyzer.