title: Illumina HiSeq 2500 sequencing; The impact of heat stress on imipenem susceptibility in imipenem resistant NTHi
design: The impact of heat stress on imipenem susceptibility in imipenem resistant NTHi
library_name: 37-SET-2_s
library_construction_protocol: The nontypeable H. influenzae (NTHi) strain GE47 examined in this study was taken from a collection of 46 NTHi strains previously studied (Cherkaoui et al., 2016 PMID: 27756711). In more detail, GE47 was isolated from the sputum of one 75 year-old patient. The inoculum suspension (50 mL) was prepared by picking several colonies from an overnight culture at 37°C on chocolate agar plates with a cotton swab and suspending the colonies in BHI broth supplemented with NAD 2 μg/mL and hemin 10 μg/mL (sBHI) to a density of 0.5 McFarland standards. The inoculum suspension was transferred in 2 sets of ten vials. Two mL of the inoculum suspension by vial. One set was then incubated at 37°C and the other at 42°C. The optical densities (D600) of 1 mL aliquots were plotted against time; and the amount of viable cells was determined at selected time points by plating culture dilutions onto chocolate agar plates, followed by overnight incubation at 37°C. The longest incubation time at 42°C that did not significantly affect cell growth was 3h. Therefore this incubation time was used in all the experiments. All culture incubations were carried out in the presence of 5% CO2. Total RNA was purified from bacterial pellets re-suspended in 1mL of TRIzol (Invitrogen) with 0.1-mm glass/zirconia beads and disrupted in a Bead Beater apparatus. The RNA in the aqueous phase was precipitated with ethanol, rinsed several times and solubilised in 50μL of diethylpyrocarbonate-treated water.  RNA libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB)
attributes.tag: Experimental Factor: temperature
attributes.value: 37