GSM5220596: 17-0604_4-delta40; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
The biofilm was washed with 100 μL phosphate-buffered saline (PBS) twice to remove planktonic cells, scraped for 2 minutes with a 200 μL pipette tip, transferred to a microcentrifuge tube, and centrifuged at 10,000 × g for 3 min. RNA was extracted using the hot phenol method. Briefly, the supernatant was removed, and pellets were resuspended in 475 μl AE buffer (50 mM sodium acetate [NaOAc], 10 mM EDTA, pH 5.2). Forty microliters of 20% SDS and 475 μl of phenol were added and incubated for 10 min at 65°C, shaking every minute. Samples were put on ice for 5 min and centrifuged for 15 min at 10,000 rpm at 4°C. Supernatants were transferred to new microcentrifuge tubes and 475 μL of chloroform was added, mixed, and centrifuged for 10 min at 2,000 rpm at 4°C. The top aqueous layer was placed in a new microcentrifuge tube, and RNA was precipitated by adding 500 μL isopropanol and 50 μL sodium acetate (2 M), centrifuging for 20 min (12,000 rpm, 4°C), and washing with 250 μL 70% cold ethanol. Finally, samples were treated with DNase (12.5% of total reaction volume, catalog number M0303L; New England BioLabs, Ipswich, MA, USA) for 45 min. RNA isolation was performed in quadruplicate.