The Drosophila brain is a work horse in neuroscience. Single-cell transcriptome analysis 1-5 , 3D morphological classification 6 , and detailed EM mapping of the connectome 7-10 have revealed an immense diversity of neuronal and glial cell types that underlie the wide array of functional and behavioral traits in the fruit fly. The identities of these cell types are controlled by -still unknowngene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. To characterize the GRN for each cell type in the Drosophila brain, we profiled chromatin accessibility of 240,919 single cells spanning nine developmental timepoints, and integrated this data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which around 70,000 are linked to specific developmental trajectories, involving neurogenesis, reprogramming and maturation. For 40 cell types, their uniquely accessible regions could be associated with their expressed transcription factors and downstream target genes, through a combination of motif discovery, network inference techniques, and deep learning. We illustrate how these "enhancer-GRNs" can be used to reveal enhancer architectures leading to a better understanding of neuronal regulatory diversity. Finally, our atlas of regulatory elements can be used to design genetic driver lines for specific cell types at specific timepoints, facilitating the characterization of brain cell types and the manipulation of brain function. MainThe brain consists of a myriad of different neuronal and glial types, each unique in their morphology and function. The Drosophila brain, which contains around 100,000 cells, is uniquely positioned as a model in which the diversity of brain cell types can be investigated. Recent advances in electron microscopy have allowed the creation of connectome maps of the different regions in the Drosophila brain 7-10 , while the availability of genetic driver lines 11 provides genetic access to many cell types for understanding neuronal function 12 . Furthermore, this diversity of cell types has been bolstered by single-cell transcriptomics on the adult brain 1-5 , the larval brain [13][14][15] , and the ventral nerve cord 16 . The recent development of single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq), makes it possible to measure chromatin accessibility of single cells in high throughput 17,18 , providing an additional crucial layer of information underlying neuronal identity: which genomic regions encode the regulatory information to create and maintain each cell type. The integrated analysis of transcriptomics and chromatin accessibility makes it then possible to jointly study enhancers and gene expression to discover precise regulatory programs across cell types [19][20][21] .Cell type identity is defined by the activity of GRNs in which combinations of transcription factors activate or repress target genes....

Single-cell RNA-seq and single-cell ATAC-seq technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost, and pave the way for more specialized multi-ome assays, custom droplet microfluidics may provide solutions complementary to commercial setups. We developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 7,996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA's high capture rate to analyse a small population of FAC-sorted neurons from the Drosophila brain, confirming the protocol's applicability to low-input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.

The lab-on-a-chip (LOC) field has witnessed an excess of new technology concepts, especially for the point-of-care (POC) applications. However, only few concepts reached the POC market often because of challenging integration with pumping and detection systems as well as with complex biological assays. Recently, a new technology termed SIMPLE was introduced as a promising POC platform due to its features of being self-powered, autonomous in liquid manipulations, cost-effective and amenable to mass production. In this paper, we improved the SIMPLE design and fabrication and demonstrated for the first time that the SIMPLE platform can be successfully integrated with biological assays by quantifying creatinine, biomarker for chronic kidney disease, in plasma samples. To validate the robustness of the SIMPLE technology, we integrated a SIMPLE-based microfluidic cartridge with colorimetric read-out system into the benchtop Creasensor. This allowed us to perform on-field validation of the Creasensor in a single-blind study with 16 plasma samples, showing excellent agreement between measured and spiked creatinine concentrations (ICC: 0.97). Moreover, the range of clinically relevant concentrations (0.76-20 mg/dL), the sample volume (5 μL) and time-to-result of only 5 min matched the Creasensor performance with both lab based and POC benchmark technologies. This study demonstrated for the first time outstanding robustness of the SIMPLE in supporting the implementation of biological assays. The SIMPLE flexibility in liquid manipulation and compatibility with different sample matrices opens up numerous opportunities for implementing more complex assays and expanding its POC applications portfolio.