RNAseq NO 2
Biofilms were cultured and dispersed as previously established  with some adjustments 33. Briefly, 107 CFU/ml bacterial solutions were prepared from overnight cultures using M9 media and 50 ml were added per tissue culture flask (Nunc, ThermoFisher Scientific). Biofilms were cultured in a shaking incubator (37oC, 70rpm) for 2, 4 or 8h. For treated groups, 100 M of SP-NONOate or 500 M of CTEMPO were added to 4h biofilms and incubated for 15 or 30 respectively. To collect planktonic cells, OD600 was measured using a spectrophotometer and approximately 108 cells were collected from the supernatant and immediately mixed with 2x volumes of RNA-protect solution (QIAGEN). The rest was discarded. To collect the biofilm cells, tissue culture flasks were rinsed with 10ml of PBS, and then 6ml of RNAprotect were mixed with 3ml of PBS and dispensed into the tissue culture flasks. Then, a cell scraper was used to detach biofilm cells from the base of the receptacle. 1ml of this suspension was used to measure the OD600. Appropriate volumes from this suspension were used to obtain up to 108 cells. Cell suspensions were subsequently pelleted at 5000 rpm (10 min, 25 oC). RNA extraction was done with the RNeasy mini kit (QIAGEN) for gram negative cells grown in minimal media. RNA purity, quantity and quality was assessed by by 260/280 and 260/230 ratios using a nanodrop device. Additionally, visual analysis of 16S and 23S bands on an agarose gel was performed by gel electrophoresis. RNA samples were DNAse treated, analysed for quality, and sequenced by BGI Genomics (Shenzhen, China).