| Construction Protocol | All bacterial cultures were incubated at 37°C with shaking at 230 rpm. The bacteria were cultivated in synthetic basal salts medium (BSM) containing 14.3 mM glucose and 0.05% casamino acids, CF serum (pooled serum samples from 5 CF patients in various stages of infection, heat inactivated at 56°C for 30 minutes) and CF sputum (pooled sputum samples from 5 CF patients of various stages of infection were diluted with BSM to a final concentration of 10% w/vol). Starter cultures were grown overnight in Luria-Bertani broth and diluted to an OD600 of 0.5. The bacteria were harvested by centrifugation and resuspended in the same volume of BSM, serum or sputum. In total, 4 ml of BSM and serum and 6.25 ml of sputum were used. The cultures were incubated for 270 minutes (37°C, 230 rpm) into the mid-log growth phase. Then, the cultures were immediately snap-cooled in liquid nitrogen and centrifuged (5 minutes, 160 x g, 4°C). The pellets were resuspended in 1 ml of Trizol (Ambion), vortexed thoroughly and incubated for 5 minutes at room temperature. Chloroform (0.2 ml) was added; the samples were shaken for 30 seconds and incubated for 3 minutes at room temperature. The samples were centrifuged (15 minutes, 160 x g, 4°C), and the upper water phase was transferred to 0.5 ml of ice-cold 70% ethanol. The RNA samples were further processed using the RiboPure-Bacteria kit (Ambion), and the MICROBExpress kit (Invitrogen) was subsequently used to remove rRNA. The quality of the mRNA was assessed using Total RNA Nano chips on a Bioanalyzer 2100 (Agilent). RNA samples were sent to the vertis Biotechnologie AG (Freising, Germany) service laboratory where cDNA preparation and library construction was performed, using TrueSeq technology (Illumina). |
FTP
