Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: After 30 minutes of growth, cells from each condition were harvested by adding 1.8 mL culture to 200 μM cold stop solution (95% ethanol, 5% acid buffered phenol, 4 °C). The mixture was centrifuged for 30 s at 13,000 rpm on a benchtop centrifuge, and the supernatant was removed with the pellet flash frozen in liquid nitrogen and stored at -80 °C. To extract RNA, Trizol (Invitrogen) was heated to 65 °C, added directly to the pellet, and incubated at 65 °C for 10 minutes with shaking at 2000 rpm (Eppendorf Thermomixer). The mixture was frozen at -80 °C for at least 10 minutes. After thawing, cells were centrifuged at 15,000 rpm, 4 °C for 5 minutes, and the supernatant was removed into 400 μL ethanol. The mixture was applied to a DirectZol spin column (Zymo) and centrifuged for 30 s at 13,000 rpm. The columns were washed with DirectZol RNA prewash buffer twice (400 μL) and RNA wash buffer (700 μL) once before eluting in 90 μL DEPC water. 10 μL 10x Turbo DNAse buffer and 2 μL Turbo DNAse (Invitrogen) were added to the eluant. The mixture was digested at 37 °C for 20 minutes, followed by the addition of 2 μL more DNAse and another 20 minute incubation. Total volume was brought to 200 mL with DEPC water and combined with 200 μL acid-phenol:chloroform (IAA, Invitrogen), vortexed and centrifuged for 10 minutes at 21,000 g and 4 °C. The top (aqueous) layer was extracted and ethanol precipitated in 20 μL NaOAc (3M), 2 μL GlycoBlue (Invitrogen) and 600 μL cold ethanol. Precipitation mix was incubated at -80 °C for more than 4 hours before centrifuging for 30 minutes at 21,000 g and 4 °C. The pellet was washed twice with 500 μL cold 70% ethanol, then air dried and resuspended in 50 μL DEPC water. RNA integrity was validated on a 6% TBE-urea acrylamide Novex gel (Invitrogen) and yield was quantified by NanoDrop spectrophotometer. rRNA was removed with the RiboZero rRNA Removal Kit for Bacteria (Illumina). RNA was fragmented and cDNA libraries were prepared using the KAPA RNA HyperPrep Kit (Roche) and sequenced on an Illumina HiSeq.