Liquid 2
Liquid growth to mid exponential phase in 1% CTMYE biorep3
For liquid cultures, a large loopful of M. xanthus DK1622 wild type was taken from a freshly grown CTTYE plate and inoculated into 25 mL 1% CTMYE in a 250 mL flask. The culture flask was shaken overnight at 32 °C and grown to a Klett measurement of approximately 80-100 and 4 mL of culture was added to 4 mL of 5% phenol in absolute ethanol. Cultures were then spun down at 8000 r.p.m. for 8 min in a Sorvall SS-34 rotor (Thermo Fisher Scientific, Waltham, MA), 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.
UC Davis
Liquid 2