Life on Earth is governed by the rhythmic rotation of the planet, dividing time into periods of light and dark to which most life forms are entrained. In mammals, time-keeping is genetically encoded by the circadian clock: a transcription-translation feedback loop lasting twenty-four hours that synchronizes cellular physiology to time-of-day. Core organismal functions are carried out under circadian regulation including even innate immunity, the body’s first line of defense against external pathogens. We set out to characterize how the brain's unique immune compartment changes around the day-night cycle via single-cell RNA sequencing (scRNAseq) of brain immune niches every six hours around a 24h clock. This web platform is intended to allow for exploration of the dataset.

To comprehensively characterize how brain immune niches change around the clock, we performed single-cell RNA sequencing (scRNAseq) of the brain and leptomeninges of healthy adult mice at Zeitgeber time points ZT0, ZT6, ZT12, and ZT18. Zeitgeber Time (ZT) refers to the number of hours elapsed from the beginning of the light cycle; this approach thus sampled around a single 24-hour light-dark cycle. Because mice are nocturnal, ZT0 and ZT6 – light cycle time points – represent the rest phase, and ZT12 and ZT18 represent the active phase.

To sensitively sample rarer immune populations including BAMs and infiltrating leukocytes, we used fluorescence-activated cell sorting (FACS) in combination with acute labeling of circulating immune cells to de-enrich microglia while still excluding peripheral contamination. To arrest cellular transcription at the time of sample collection, prevent dissociation-induced gene signatures, and ensure transcriptional profiles were faithful to collection time, samples were treated with validated transcriptional and translational inhibitors throughout the collection pipeline.