Density gradient formation and RNA distribution. (a) Buoyant densities (BD) of gradient fractions averaged over 12 gradients. The standard error of the mean (SEM) for each fraction was ≤0.0045 g mL−1
. Vertical dash-dot lines classify the division of the gradients into “heavy” (fractions 1–5; BD 1.84–1.807 g mL−1
), “medium” (fractions 6–10; BD 1.801–1.781 g mL−1
), and “light” RNA fractions (fractions 11–16; BD 1.777–1.743 g mL−1
). (b) Density-dependent RNA concentration in gradient fractions of the 40 mM [U13
C]glucose cultures and the uncultured control. RNA was isolated from mice faeces at the start of the incubation (0 h control), after 2 h, and after 4 h from the [U13
C] in vitro cultures, and resolved in a density gradient solution by ultracentrifugation. Separated RNA was harvested and quantified with a RiboGreen low range assay. To facilitate comparison between the gradients, the RNA content is given in relative units (%; fraction with the highest RNA concentration per gradient was set as 100%) [23
]. Arrows indicate the gradient fractions, which were chosen for further downstream analysis by NGS.