title: Solid growth for four days on 1% CTMYE agar biorep2
design: For solid cultures, 1% CTMYE plates (1% agar) were streaked with M. xanthus DK1622 wild type from a frozen stock and incubated for approximately 4 days at 32 °C. One large loopful was taken and resuspended in 0.75 mL MC7 buffer and added to 0.75 mL 5% phenol in absolute ethanol. Cultures were then spun down at 8000 r.p.m. for 3 min in a microcentrifuge, supernatant was poured off, and pellets were snap-frozen in liquid N2.     Total cellular RNA was extracted from N2 snap-frozen M. xanthus cells using the RNeasy mini kit (Qiagen, Valencia, CA) for three batch liquid, three solid plate samples, and two flow cell samples. RNA concentrations were determined from measurements on a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) at A260. To each sample, 1 µL of 50 pg/µL firefly luciferase (Fluc) mRNA, generated by T7 RNA synthesis (New England Biolabs, Ipswich, MA), was added as an absolute concentration control. Approximately 400 ng of total cellular RNA of each sample was used to generate RNAseq libraries using the Encore complete prokaryotic RNAseq DR multiplex system (NuGEN, San Carlos, CA) exactly as described in the manufacturer’s protocol.