CdnLDD60-G_rep3
Cultures of 3 independent colonies of EG865 and EG2530 were inoculated into 2 mL M2G overnight. The next day, cultures were diluted into 4 mL M2G and grown to an OD600 of 0.4 0.6. Cells were prepared for carbon starvation as described above, except after the final wash, cells were resuspended in 4 mL M2 and were split into two 2 mL samples each. One sample for each replicate was incubated at 30C shaking at 225 rpm for 60 minutes, while the other was stabilized using RNAprotect Bacteria Reagent (Qiagen Catalog #76506) following manufacturers instructions. Briefly, 4 mL RNAprotect was added to 15 mL conical tubes, to which the 2 mL culture samples were added. The conical tubes were vortexed for 5 seconds, incubated at room temperature for 5 minutes, and then centrifuged for 10 minutes at 5,000 x g. The pellets were flash frozen in liquid nitrogen and stored at -80C. The 60-minute starved samples were harvested in the same way. All samples were processed at SeqCenter (Pittsburg,PA). There, samples were DNAse treated with Invitrogen DNAse (RNAse free). Library preparation was performed using Illuminas Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and 10bp IDT for Illumina indices and Caulobacter-specific rRNA depletion probes. Sequencing was done on a NextSeq2000 giving 2x51bp reads.