title: Flow cell growth for four days in 1% CTMYE biorep1
design: For flow cell cultures, single chamber flow cells were assembled as described above and filled with 1% CTMYE + 4 μg/mL gentamycin. A large loopful of M. xanthus DK1622 wild type was taken from a freshly grown CTTYE plate and inoculated into 25 mL 1% CTMYE + 4 μg/mL gentamycin 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 2 mL was spun down at 8000 r.p.m. for 3 min in a microcentrifuge, supernatant was poured off, and pellets were resuspended in 2 mL MC7 buffer. 0.75 mL of culture was then injected via sterile 3 mL syringe with a 25 x 5/8” needle into the flow cell through the tubing attached to the input port. Bacterial attachment was allowed to occur for at least 2 hours and the flow was turned on to a setting of 2.5 on the Rainin model RP-1 peristaltic pump (Rainin Instrument LLC, Oakland, CA). Biofilms were grown statically under continuous flow at 32 °C for 4 days in 1% CTMYE + 4 μg/mL gentamycin. Coverslips supporting the growth of the biofilms were removed and placed into a 50 mL Falcon tube containing 0.5 mL MC7 buffer and 0.5 mL 5% phenol in absolute ethanol. Cells were scraped off the coverslip using a pipette tip and were resuspended via vortexing. Cultures were placed into a 1.5 mL microfuge tube, spun down at 8000 r.p.m. for 3 min in a microcentrifuge, supernatant was aspirated 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.