GSM8636656: Bfrag-S4; Bacteroides fragilis 638R; RNA-Seq
Cells were flash-frozen in a dry ice-ethanol slurry, then pelleted by centrifugation at 16,000xg for 1 minute at 4°C. The bacterial pellets were stored at −80°C until RNA extraction. RNA was extracted using TRIzol reagent and the PureLink™ RNA Mini Kit following the manufacturer's instructions. Briefly, bacterial pellets were incubated with 1 mL of TRIzol for 5 minutes at room temperature, followed by the addition of 200 µL of chloroform. After 5 minutes at room temperature, the samples were centrifuged at 12,000xg for 15 minutes at 4°C. The aqueous phase was transferred to a new tube, and an equal volume of 100% ethanol was added. This mixture was transferred to a spin cartridge and centrifuged at 12,000xg for 15 seconds at room temperature. RNA samples were treated with PureLink™ DNase I, according to the manufacturer's protocol. Following DNase treatment and four wash steps, RNA was eluted in 50 µL of RNase-free water by centrifugation at 12,000xg for 1 minute. Eluted RNA was transferred to RNase-free tubes and analyzed for quality by agarose gel electrophoresis. RNA integrity was assessed using the Bio-Rad ChemiDoc system, and RNA concentration was quantified with a NanoDrop One spectrophotometer (Thermo Fisher Scientific). Further RNA quality and concentration was validated on an Agilent 4200 TapeStation RNA ScreenTape, with all RIN scores above 8.0 and samples normalized to 250 ng starting mass. Library preparation was performed using Illumina's Stranded Total RNA Prep with Ribo-Zero Plus Microbiome, which targets depletion of microbiome rRNA prior to cDNA synthesis and library formation. Final libraries were validated using an Agilent 4200 TapeStation D1000 ScreenTape, with an average fragment size of 396bp. Libraries were quantified using Invitrogen's Quant iT High Sensitivity dsDNA Kit and normalized during pooling.