Genome-wide c-di-GMP targets in Streptomyces

Cell material from three S. venezuelae macro colonies grown on MYM-agar for 30 hours at 30C was pooled and then resuspended in 200 ul ice-cold stop solution. After centrifugation, the supernatant was removed and the cell pellets were stored at -80C until RNA isolation.Cell pellets were resuspended in 700 ul RLA Buffer (Promega) and then split into two 350 ul fractions while one fraction was used immediately and the other one was stored again at -80C. After the addition of 352 ul Phenol (Roth, RNA, pH 4,3) and 88 ul Chloroform-Isoamylalcohol (24:1, AppliChem). Homogenization was performed using a BeadBlaster 24 (Benchmark). Three pulses of 30 seconds of intensity 6.0 m/s were applied with cooling down for 1 minutes between pulses. RNA quantity and quality was analyzed using NanoDrop 2000 (Thermo Scientific) and Bioanalyzer 2100 (Agilent) measurements. rRNA was depleted and libraries were made by vertis Biotechnologie AG.