Extended Data Fig. 1. Schematic detailing the BOSSS methodology used to identify dietary cholesterol-interacting microbes and their metabolic transformations of cholesterol.

a, An alkyne-modified form of cholesterol is orally introduced to mice which allows for the tracking of dietary cholesterol and derivatives of dietary cholesterol into the microbiome of mice. Isolated microbiome samples from caecal contents are b, sorted to separate interacting (Alk+) versus non-interacting (Alk-) microbes in order to c, identify these microbes using metagenomic sequencing. d, Separately, metabolomes from unsorted microbiome samples from cholesterol-alkyne exposed mice can be compared to metabolomes from mice exposed to native cholesterol to determine differential features found in the cholesterol alkyne exposed state. These features are then queried in the native cholesterol state to make sure that the transformation is not an artifact of the alkyne modification of cholesterol.