Abstract: The disclosure provides methods and systems of analyzing single cells by simultaneously separating cells into monodisperse droplets and tagging each nucleic acid molecule from the cells with barcodes unique to each droplet. The methods and systems combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets encapsulating a single one of the template particles and a single one of the single cells, release nucleic acid molecules from the single cells and provide each nucleic acid molecule with a barcode unique to the respective droplet. The nucleic acid molecules can then be analyzed by any known method, for example by sequencing the nucleic acid molecules.

Abstract: The invention provides a system for evaluating gene expression levels from scRNA-Seq experiments. The system uses in silico cell calling tools for distinguishing between partitions that contain cells and background partitions that did not fully capture a cell. The system also provides barcode processing tools that tests barcodes from at least cellular and UMI tiers against a whitelist and optionally converts those to a compact index format. Further, the system can implement multimapping tools to preserve information for reads that map to multiple locations in a reference.

Abstract: This disclosure provides methods and systems for single-cell analysis, including single-cell transcriptome analysis, of target cells without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target cell from a population of cells in an encapsulation, derive a plurality of distinct mRNA molecules from the single target cell, and quantify the distinct mRNA molecules to generate an expression profile.

Abstract: The disclosure provides methods and systems of analyzing single cells by simultaneously separating cells into monodisperse droplets and tagging each nucleic acid molecule from the cells with barcodes unique to each droplet. The methods and systems combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets encapsulating a single one of the template particles and a single one of the single cells, release nucleic acid molecules from the single cells and provide each nucleic acid molecule with a barcode unique to the respective droplet. The nucleic acid molecules can then be analyzed by any known method, for example by sequencing the nucleic acid molecules.

Abstract: The invention provides methods and systems for drug screening by segregating single cells into droplets simultaneously and providing candidate compound to the single cells to measure cellular response. Methods of the present invention combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells, provide to the single cells one or more candidate compounds, and measure a cellular response to the one or more candidate compounds.

Abstract: This disclosure provides a decentralized workflow for analyzing single cell gene expression. The workflow makes use of pre-templated instant partitions to segregate cells into separate compartments to individually capture and barcode RNA from single cells in a massively parallel single tube format. The workflow includes steps for processing the RNA from the single cells for sequencing. Separate portions of the decentralized workflow are performed by a research lab and a core facility, allowing increased flexibility in time and location of protocol steps.

Abstract: This invention provides methods for near-instantaneously separating cells that have undergone RNA guided genome modifications into pre-templated instant partitions (PIPs) and using the PIPs to associate the guide RNAs with the gene expression level changes that resulted from the genome modification.

Abstract: The invention provides methods and systems for drug screening by segregating single cells into droplets simultaneously and providing candidate compound to the single cells to measure cellular response. Methods of the present invention combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells, provide to the single cells one or more candidate compounds, and measure a cellular response to the one or more candidate compounds.

Abstract: The invention provides high-throughput systems and methods for screening CRISPR-edited cells in bulk with single cell resolution. Methods of the invention use cells expressing polyadenylated guide RNAs that are detectable by RNA sequencing. Methods of the invention provide for the detection of each cell's guide RNA along with its single cell transcriptome to provide useful gene expression data for assessing CRISPR activity from cells in bulk. In addition, methods of the invention offer a high throughput single cell analytical framework for generating single cell transcriptome data from which CRISPR activity may be evaluated.

Abstract: This disclosure provides methods and systems for single-extracellular (EV), multi-omic analysis of target EVs without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target EV from a population of EVs in an encapsulation, derive a plurality of distinct mRNA molecules from the single target EV, and quantify the distinct mRNA molecules to generate an expression profile. Nucleic-acid-tagged antibody conjugates are used for simultaneous proteomic analysis along with the gene expression profiling, which enables classification of an EV in a sample.

Abstract: Methods to emulsify cells and/or mRNA with reverse transcriptase at a temperature such that the reverse transcriptase begins making cDNA during the emulsification

Abstract: This disclosure provides methods and systems for single-cell analysis, including single-cell transcriptome analysis, of target cells without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target cell from a population of cells in an encapsulation, derive a plurality of distinct mRNA molecules from the single target cell, and quantify the distinct mRNA molecules to generate an expression profile.

Abstract: The disclosure provides methods for creating long oligonucleotide reagents that include barcodes and other element for sequencing library preparation, where the oligonucleotides are created by multiple tiers of ligation of shorter oligos. The disclosed methods work to extend short oligos that are attached to particles, thereby allowing one to create particles that carry large number of long sample preparation oligonucleotides without being required to synthesize those full-length molecules with a polymerase.

Abstract: The disclosure provides methods and systems of analyzing single cells by simultaneously separating cells into monodisperse droplets and tagging each nucleic acid molecule from the cells with barcodes unique to each droplet. The methods and systems combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets encapsulating a single one of the template particles and a single one of the single cells, release nucleic acid molecules from the single cells and provide each nucleic acid molecule with a barcode unique to the respective droplet. The nucleic acid molecules can then be analyzed by any known method, for example by sequencing the nucleic acid molecules.

Abstract: The methods and systems described herein provide capture template particles and an improved emulsion droplet-based target capture and barcoding method thereof. The capture template particles and methods disclosed herein allow capturing targets of interest from biological samples, and barcoding of specific nucleic acids contained in the captured targets. The nucleic acids can be contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA.

Abstract: The disclosure provides methods and systems of analyzing single cells by simultaneously separating cells into monodisperse droplets and tagging each nucleic acid molecule from the cells with barcodes unique to each droplet. The methods and systems combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets encapsulating a single one of the template particles and a single one of the single cells, release nucleic acid molecules from the single cells and provide each nucleic acid molecule with a barcode unique to the respective droplet. The nucleic acid molecules can then be analyzed by any known method, for example by sequencing the nucleic acid molecules.

Abstract: The disclosure provides methods and systems of analyzing single cells by simultaneously separating cells into monodisperse droplets and tagging each nucleic acid molecule from the cells with barcodes unique to each droplet. The methods and systems combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets encapsulating a single one of the template particles and a single one of the single cells, release nucleic acid molecules from the single cells and provide each nucleic acid molecule with a barcode unique to the respective droplet. The nucleic acid molecules can then be analyzed by any known method, for example by sequencing the nucleic acid molecules.

Abstract: The invention provides devices that generate monodisperse droplets from a bulk liquid. The devices include a shearing mechanism, a holder for a vessel containing a liquid, and an optical system that transmit light to, and detects light from, liquid in the vessel. The invention also provides methods of using such devices to produce monodisperse droplets.

Abstract: This disclosure provides methods and systems for amplifying small amounts of nucleic acids. Methods include performing amplification reactions in an emulsion format to isolate and clonally amplify discrete populations of nucleic acid molecules inside droplets. In particular, methods and systems of the invention generate an emulsion with particles that template the formation of droplets inside a tube and segregate nucleic acid molecules therein such that each droplet contains an individual nucleic acid molecule. The nucleic acid molecules are amplified inside the droplets.

Abstract: The invention provides methods and systems for drug screening by segregating single cells into droplets simultaneously and providing candidate compound to the single cells to measure cellular response. Methods of the present invention combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells, provide to the single cells one or more candidate compounds, and measure a cellular response to the one or more candidate compounds.