Pathway and gene-set enrichment analysis revealed hierarchical associations between fibroblast subpopulations that were consistent with transcriptomic similarities, and also suggested heterogeneous pathway activation between these groups (Fig.?4d). clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad power in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques. Introduction The complex architecture and associated higher-order function of human tissues relies on functionally and molecularly diverse cell populations. Disease says represent significant perturbations to cellular heterogeneity, with tissue-resident cells acquiring altered phenotypes and circulating cells infiltrating into the tissue. Therefore, defining the cellular subsets found in pathologic tissues provides insights into disease etiology and treatment options. Traditional methods such as flow cytometry, which require a priori knowledge of cell type-specific markers, have begun to define this scenery, but fall short in comprehensively identifying cellular states in a tissue, with particular difficulty detecting extremely rare subpopulations. Technological advancements in automation, microfluidics, and molecular barcoding schemes have permitted the sequencing of single cells with unprecedented throughput and resolution1C4. In particular, recent studies featuring analysis of 104C105 single cells have enabled unbiased profiling of cellular heterogeneity, GLPG0974 where entire tissues can be profiled without GLPG0974 advance enrichment of individual cell types1,5,6. In spite of this progress, technological advances can be slow to permeate into resource-limited clinical arenas GLPG0974 due to a variety of reasons related to cost, personnel requirements, space or infrastructure. Specifically, a major barrier to widespread adoption of droplet microfluidic techniques is the lack of cost-effective and reliable instrumentation7,8. Microfluidic experiments are typically performed using commercial instruments which are expensive and often configured for a single purpose, or custom research instrument setups which are comprised of multiple pieces of equipment and rarely portable. Particularly in clinical settings, microfluidic instrumentation is not usually proximal to the site of cell sample generation requiring transport to external sites or cell preservation, both of which can alter cellular transcriptomes or result in extensive cell death6,9. To address these short-comings and provide a low-cost option for single-cell transcriptome profiling, we have developed an GLPG0974 open-source portable instrument for performing single-cell droplet microfluidic experiments in research and clinical settings. Recent microwell-based transcriptome profiling approaches have been shown to be advantageous for low-cost portable transcriptome profiling10C12, however some of these techniques are challenging to perform and or require extensive chemical modification to fabricate the devices. Additionally, the fixed architecture of microwell (partitioning) microfluidic devices dictates their use PDGFRA for specific applications. In contrast, the platform presented here is easy to use and can be implemented for a variety of droplet microfluidic (partitioning) or continuous phase microfluidic based experiments. Potential applications of this system include recent work profiling immune repertoires from hundreds of thousands of single cells13 and combined single-cell transcriptome and epitope profiling14 in addition to ddPCR15, ddMDA16, hydrogel microsphere fabrication for 3D cell culture17,18, chemical microfluidic gradient generation19 and microparticle size sorting20C22. The instrument is usually comprised of electronic and pneumatic components affixed to a 3D printed frame. The entire system is usually operated through software control using a graphical GLPG0974 user interface on a touchscreen. Requiring only a standard wall power outlet, the instrument has an extremely small footprint; small enough to fit on a bench top or in a biocontainment hood. The total cost of materials to construct an instrument is usually approximately $575. This represents an approximately 20-fold?and 200-fold reduction in cost?compared to a research-level, syringe-pump based microfluidic setup, and a commercial microfluidic platform,?respectively. We applied the microfluidic control instrument in conjunction with the Drop-seq technique1 to perform unbiased identification of transcriptomic says in diseased synovial tissue, which becomes highly inflamed in rheumatoid arthritis (RA) and drives joint dysfunction. RA is usually a common autoimmune disease affecting approximately 1% of the population. While the cause of RA is not precisely known, disease etiology is usually hypothesized to originate from a combination of environmental and genetic factors23,24. RA affects the lining of the joint; the synovial membrane, leading to.
