Rnalater solution

On experiment days 1–5, the animals were injected IP with DEX or vehicle. On experiment day 5 between 16:00–18:00, animals were deeply anesthetized (mice with a ketamine-xylazine-acepromazine cocktail, rats with 4% isofluorane), decapitated, and whole brains removed. This time point coincides with the expected orexin nadir.[10 (link)] For mouse in situ hybridization, whole brains were frozen on dry ice and stored at -80°C until the time of assay. For mice and rat hypothalamus mRNA expression and for rat hypothalamus quantitative orexin protein analysis, hypothalamic blocks (including the hypothalamus, most diencephalic structures, and the prefrontal cortex) were excised from the whole brain as described previously.[32 (link)] Blocks were preserved in RNAlater solution (Ambion), stored at 4°C overnight, then frozen at -80°C without RNAlater solution until the time of assay.

Planktonic cells from the surrounding medium were transferred to centrifuge tubes with O-ring seals under anaerobic conditions in the anaerobic chamber and collected by centrifuging at room temperature (6,000 ×g). To keep their gene expression profiles intact, the planktonic cells were put into a RNALater solution immediately (Ambion, Carlsbad, CA, USA) (Bachoon et al., 2001 (link); Mason et al., 2012 (link); Shi et al., 2013 (link); Qi et al., 2014 (link)).
For the D. vulgaris biofilms, superficial cells of the mature biofilm formed on the surface of SS slides were slightly washed off with 50 mM oxygen-free phosphate-buffered saline (PBS, pH 6.5) using a dropper under anaerobic conditions in the anaerobic chamber, then inner mature biofilm cells were scraped using a sterile razor blade from the surface of SS slides. To keep their gene expression profiles intact, the biofilm cells were also put into a RNALater solution immediately (Ambion, Carlsbad, CA, USA) (Zhang et al., 2007 (link)). The suspension of biofilm cells was then heavily vortexed to release single D. vulgaris cells to be used for single-cell isolation (Chalmers et al., 2007 (link); Marcy et al., 2007 (link); Zhang et al., 2007 (link)).

After collection, the biopsy fragments were immediately placed into an RNAlater solution (Applied Biosystems™, USA) and stored at -20 °C until use. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germany), and RNA concentration and quality were measured using the NanoDrop 2000 Spectrophotometer (NanoDrop, USA). The concentration was adjusted to 500 ng, and cDNA synthesis was conducted using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems™, USA) following the manufacturer’s instructions.