GSM4890888: Mut5_1; Bacillus amyloliquefaciens; RNA-Seq
Single colonies of PCL1606 and FZB42 WT and the GlpK mutant were grown overnight in LB medium at 30°C. Overnight cultures were diluted to a starting OD600 of 0.01 in the same medium and 2 µl were spotted on King B plates alone and in interaction between PCL1606 and the Bacillus strains. Cultures were grown for 24h at 30°C. After that, colonies were recovered and stored at -80C. The collected cells were resuspended and homogenized by passing through a 25 5/8 G needle in BirnBoim A (20% sucrose, 10mM tris-HCl pH 8, 10mM EDTA and 50mM NaCl). Lysozyme (10 mg/ml) was added and incubated 30 min at 37C. After disruption, suspensions were centrifuged, and pellets were resuspended in Trizol reagent (Invitrogen). Total RNA extraction was performed as indicated by the manufacturer. Removal of DNA was carried out by in-column treatment with the rDNAse included in the Nucleo-Spin RNA Plant (Macherey-Nagel) following instructions from the manufacturer. Integrity and quality of total RNA was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies) and by gel electrophoresis. RNA libraries were prepared for sequencing using standard Illumina protocols