Highlights d Exposure to the psychedelic drug DOI results in enduring molecular adaptations d Post-acute DOI unveils phenotypes akin to antidepressant adaptations d Concurrent occurrence of synaptic plasticity mediated via 5-HT

The inherent heterogeneity in cell populations has become of great interest and importance as analytical techniques have improved over the past decades. With the advent of personalized medicine, understanding the impact of this heterogeneity has become an important challenge for the research community. Many different microfluidic approaches with varying levels of throughput and resolution exist to study single cell activity. In this review, we take a broad view of the recent microfluidic developments in single cell analysis based on microwell, microchamber, and droplet platforms. We cover physical, chemical, and molecular biology approaches for cellular and molecular analysis including newly emerging genome-wide analysis.

Epigenetic mechanisms such as histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling various histone modifications at the genome scale using tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a cell type, cell isolation from tissues is often necessary to generate a homogeneous cell population and such operation tends to yield a low number of cells. In addition, high-throughput processing is often desirable due to the multiplexity of histone marks of interest and the large quantity of samples in a hospital setting. In this protocol, we describe detailed instructions on device fabrication, setup, and operation of microfluidic oscillatory washing-based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling histone modifications using as few as 100 cells per assay with a throughput as high as 8 assays in a run. MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads followed by vigorous microfluidic oscillatory washing. Our process is semi-automated for reduced labor and improved reproducibility. Using one 8-unit device, it takes 2 d to produce 8 sequencing libraries from chromatin samples. The technology is scalable. We used the protocol to study a number of histone modifications in various types of mouse and human tissues. The protocol can be conducted by a user who is familiar with molecular biology procedures and has basic engineering skills.

Emerging studies suggest that monocytes can be trained by bacterial endotoxin to adopt distinct memory states ranging from low-grade inflammation to immune exhaustion. While low-grade inflammation may contribute to the pathogenesis of chronic diseases, exhausted monocytes with pathogenic and immune-suppressive characteristics may underlie the pathogenesis of polymicrobial sepsis including COVID-19. However, detailed processes by which the dynamic adaption of monocytes occur remain poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low-dose or high-dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, high dose LPS uniquely upregulates exhaustion signatures with metabolic and proliferative pathways. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies.