Project description:Neutrophils play critical roles in modulating the immune response. However, neutrophils have a short circulating half life, are readily stimulated in vitro, and have low levels of cellular mRNA when compared to other blood leukocyte populations. All of these factors have made it difficult to evaluate neutrophils from clinical populations for molecular and functional studies. Here we present a robust methodology for rapidly isolating neutrophils directly from whole blood and develop âon- chipâ processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and demonstrate the ability of the device to discriminate subtle differences in the genomic and proteomic response of peripheral blood neutrophils to direct and indirect stimulation. Lastly, we implement this tool as part of a near patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury and demonstrate that this technique is easy to use by nurses and technical staff yielding excellent quality and sufficient quantity of mRNA for sensitive genomic readout of the host response to injury 1. Ex vivo Stimulation Studies: We assessed whether the relatively small number of isolated neutrophils captured by the microfluidics cassettes would impact the resulting genomic sensitivity and potential discriminatory genomic capabilities in response to various stimuli. To do this, we compared the genome-wide expression profile in neutrophils from 4 independent repeated experiements under three conditions - untimulated, ex vivo activation with either Escherichia coli lipopolysaccharide (LPS), or with granulocyte-macrophage colony-simulating factor (GM-CSF) and interferon-gamma (INF-g) (referred to as GM+I). In both protocols, whole blood was stimulated ex vivo24 to allow leukocyte and plasma protein interactions. 2. Inter-subject Reproducibility: To further ensure the reliability of the of the microfluidics cassette isolation method, we directly compared the gene expression of neutrophils captured in the microfluidics cassettes with neutrophils isolated using density centrifugation with Ficoll-dextran. We performed parallel neutrophil isolations using both methodologies from five different healthy volunteers and processed the cell lysates for microarray analysis using identical protocols.
Project description:Inflammation is a realized detriment to brain health in a growing number of neurological diseases, but querying neuroinflammation in its cellular complexity remains a challenge. This manuscript aims to provide a reliable and accessible strategy for examining the brain’s immune system. We compare the efficacy of cell isolation methods in producing ample and pure immune samples from mouse brains. Then, with the high-input single-cell genomics platform PIPseq, we generate a rich neuroimmune dataset containing microglia and many peripheral immune populations. To demonstrate this strategy’s utility, we interrogate the well-established model of LPS-induced neuroinflammation with single-cell resolution. We demonstrate the activation of crosstalk between microglia and peripheral phagocytes and highlight the unique contributions of microglia and peripheral immune cells to neuroinflammation. Our approach enables the high-depth evaluation of inflammation in longstanding rodent models of neurological disease to reveal novel insight into the contributions of the immune system to brain health.
Project description:Single-cell multi-omics are powerful means to study cell-to-cell heterogeneity. Here, we present a single-tube, bisulfite-free method for the simultaneous, genome-wide analysis of DNA methylation and genetic variants in single cells: epigenomics and genomics of single cells analyzed by restriction (epi-gSCAR). By applying this method, we obtained DNA methylation measurements of up to 506,063 CpGs and up to 1,244,188 single-nucleotide variants from single leukemia-derived cells. We demonstrate that epi-gSCAR generates accurate and reproducible measurements of DNA methylation and allows to differentiate between two cell lines based on the DNA methylation and genetic profiles.
Project description:The compartmentalization of reactions in monodispersed droplets is valuable for applications across biology. However, the requirement of microfluidics to partition the sample into monodispersed droplets is a significant barrier that impedes implementation. Here, we introduce particle-templated emulsification, a method to encapsulate samples in monodispersed emulsions without microfluidics. By vortexing a mixture of hydrogel particles and sample solution, we encapsulate the sample in monodispersed emulsions that are useful for most droplet applications. We illustrate the method with ddPCR and single cell culture. The ability to encapsulate samples in monodispersed droplets without microfluidics should facilitate the implementation of compartmentalized reactions in biology.
Project description:Integrated single-cell transcriptome and DNA methylome profiling has provided insight into the complex regulatory networks of cells. Existing methods are based on picking a single-cell and performing library construction in a tube, which is costly and cumbersome. Here, we propose DIRECT, a digital microfluidics-based method for simultaneous analysis of the methylome and transcriptome in a single library construction. The accuracy of DIRECT is demonstrated in comparison with bulk and single-omics data, and the high CpG site coverage of DIRECT allows for precise analysis of copy number variation information, enabling expansion of single cell analysis from two- to three-omics. By applying DIRECT to monitor the dynamics of mouse embryonic stem cell differentiation, the relationship between DNA methylation and changes in gene expression during differentiation was revealed. DIRECT enables accurate, robust, and reproducible single-cell DNA methylation and gene expression co-analysis at a lower cost and with greater efficiency, broadening the application scenarios of single-cell multi-omics analysis and revealing a more comprehensive and fine-grained map of cellular regulatory landscapes.
Project description:While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up LC-MS workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we here established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40% v/v formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analysis of single Caenorhabditis elegans nematodes, increasing the number of proteoform identifications compared to in-tube sample preparation by 46%. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in abundance of proteoforms not distinguishable by bottom-up proteomics. The workflow presented here will help to elucidate cellular changes on the proteoform level in other samples of limited availability.