Abstract: The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. The invention provides for aqueous based emulsions containing uniquely labeled cells, enzymes, nucleic acids, etc., wherein the emulsions further comprise primers, labels, probes, and other reactants. An oil based carrier-fluid envelopes the emulsion library on a microfluidic device, such that a continuous channel provides for flow of the immiscible fluids, to accomplish pooling, coalescing, mixing, sorting, detection, etc., of the emulsion library.

Abstract: Provided herein are compositions, kits, and methods for collection, integration and analysis of various facets of information from tissues at the cellular or subcellular level. Information includes spatial mapping from nucleic acid barcodes to reconstruct location of nodes of nucleic acid barcode generation in a sample. In some workflows, light-based technologies are incorporated for an additional layer of selective spatial tagging of regions. In further steps, such tags are optionally analyzed by high throughput imaging or Next Generation Sequencing.

Abstract: The present invention is generally related to systems and methods for producing a plurality of droplets. The droplets may contain varying species, e.g., for use as a library. In some cases, the fluidic droplets may be rigidified to form rigidified droplets (e.g., gel droplets). In certain embodiments, the droplets may undergo a phase change (e.g., from rigidified droplets to fluidized droplets), as discussed more herein. In some cases, a species may be added internally to a droplet by exposing the droplet to a fluid comprising a plurality of species.

Abstract: The present invention pertains to a novel method for the generation of highly diverse RNA expressing vectors and vector libraries for use in targeted gene knock out, knock down and genome modification approaches. The invention pertains to a method for generating such higher order libraries without the need of classical cloning technologies. This is particularly useful for libraries based on large vectors wherein a sequence cannot be easily mutated with classical mutagenesis methods. The vectors and libraries generated according to the methods of the invention are in particular for RNA assisted silencing technologies such as RNA interference, and for targeted genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system or similar RNA/DNA-encoded gene perturbation systems which use small guide RNAs to target the CRISPR complex to a specific genomic sequence. The invention provides also kits comprising the materials for performing the methods of the invention.

Abstract: Systems, kits, and methods for detecting and quantifying proteomic activity using DNA-encoded probes are provided, where the proteomic activity may be enzymatic activity or ligand binding affinity. Such systems and methods encode quantitative proteomic activity information into DNA sequence populations and utilize DNA-linked substrates or ligands as activity probes. The systems, kits, and methods that are directed to detecting ligand affinity further include crosslinking steps to ensure the integrity of the DNA-linked ligands during purification and washing. Signal detection involves the chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for desired products. Selection-induced changes in DNA abundance between the initial pool and the purified pool indicate sample activity.

Abstract: Transcriptomes of individual neurons provide rich information about cell types and dynamic states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation from dense adult tissue is challenging, and markers for each phase are limited. Here, Applicants developed Nuc-seq, Div-Seq, and Dronc-Seq. Div-seq combines Nuc-Seq, a scalable single nucleus RNA-Seq method, with EdU-mediated labeling of proliferating cells. Nuc-Seq can sensitively identify closely related cell types within the adult hippocampus. Div-Seq can track transcriptional dynamics of newborn neurons in an adult neurogenic region in the hippocampus. Dronc-Seq uses a microfluidic device to co-encapsulate individual nuclei in reverse emulsion aqueous droplets in an oil medium together with one uniquely barcoded mRNA-capture bead. Finally, Applicants found rare adult newborn GABAergic neurons in the spinal cord, a non-canonical neurogenic region.

Abstract: Methods of double-stranded nucleic acid sequence determination and assembly that are able to identify insertions, deletions, repeat region sizes and genomic rearrangements, for example, are disclosed herein, which can use relatively large labeled nucleic acid fragments to analyze the structure of even larger genetic regions. In some embodiments these methods involve the use of certain parameters which unexpectedly improve overall method performance. In some embodiments these methods involve sample labeling that does not result in the formation of single-stranded nucleic acid fragment labeling intermediaries.

Abstract: Provided herein are methods for screening biological functions of microscale biological systems comprises segregating each microscale biological system from a set of microscale biological systems to be screened into individual discrete volumes, the individual discrete volume comprising a first polymer. The first polymer is then forced or allowed to polymerize to form a set of polymerized beads that encapsulate an individual microscale biological system. The polymerized beads are further encapsulated in a second droplet comprising a second polymer and one or more reporter elements. The reporter elements are configured to produce a readout upon detecting the absence or presence of a biological function to be screened. The second polymer is then forced or allowed to polymerize to form an outer capsule around each individual bead thereby forming a set of encapsulated beads. One or more biological functions of the double-encapsulated system are identified by detecting the readout of the reporters.

Abstract: Disclosed herein are compositions, methods and kits useful for epigenetic analysis based on the use of transposons that are targeted to specific regions of chromatin based on DNA-DNA interactions, protein-protein interactions, RNA-RNA interactions, and nucleic acid-protein interactions.

Abstract: Provided herein are methods for target gene sequencing and single cell barcoding in conjunction with analysis of gene expression in single cells. In some embodiments, the target gene is an immune molecule, such as an antibody or TCR. In some embodiments, the methods can be used to carry out transcriptome sequencing, e.g., RNA sequencing, to capture transcriptome of single cells paired with full receptor immune receptor sequences such that information about the immune repertoire and transcriptome of a cell can be determined. Also provided are polynucleotide libraries for use in carrying out transcriptome analysis and immune molecule, e.g., antibody or TCR, sequencing.

Abstract: In accordance with embodiments herein a method for capturing cells of interest in a digital microfluidic system is provided, comprising utilizing a droplet actuator to transport a sample droplet to a microwell device. The microwell device includes a substrate having a plurality of microwells that open onto a droplet operations surface of the microwell device. The sample droplet includes cells of interest that enter the microwells. The method introduces capture beads to the microwells, and the capture elements are immobilized on the capture beads. The method utilizes the droplet actuator to transport a cell lysis reagent droplet to the microwell device. Portions of the cell lysis reagent droplet enter the microwells and, during an incubation period, cause the cells of interest to release analyte that is captured by the capture elements on the capture beads.

Abstract: Contiguity information is important to achieving high-quality de novo assembly of mammalian genomes and the haplotype-resolved resequencing of human genomes. The methods described herein pursue cost-effective, massively parallel capture of contiguity information at different scales.

Abstract: A parallelized chain-synthesizing technique includes capillary tubes, where each tube provides multiple locations or addresses where a specific arbitrary sequence for polymeric chains can be synthesized. An optical addressing system selectively delivers light to the locations to mediate or control reactions in the tubes.

Abstract: Provided herein are methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells. In one embodiment, the methods include linear amplification of the nucleic acids. In one embodiment, the sequencing library includes whole genome nucleic acids from the plurality of single cells. In one embodiment, the nucleic acids include three index sequences. Also provided herein are compositions, such as compositions that include the nucleic acids having three index sequences.

Abstract: The present invention is related to a method for recovering two or more genes, or gene products, or cDNAs, encoding for an immunoreceptor having two or more subunits, which two or more genes, or gene products, are comprised in a given source cell. The invention is further related to a method of creating a library of expressor cells, in which library each cell is capable of expressing two or more genes, or gene products, encoding for the subunits of the immunoreceptor. The invention is further related to a method of screening a library of expressor cells as created according to the above method, for one cell that expresses an immunoreceptor that has specificity for a given target molecule.

Abstract: The disclosed methods and kits are useful in processing and analyzing a multiplicity of samples in molecular biology workflows where there is an increased chance for sample cross-contamination or misidentification. Some embodiments of the methods and kits utilize at least one spike in control and at least one barcode per sample.

Abstract: Methods and compositions are described for single cell resolution, quantitative proteomic analysis using high throughput sequencing.

Abstract: In one aspect, the present invention relates to a method for obtaining structural information about an encoded molecule. The encoded molecule may be produced by a reaction of a plurality of chemical entities and may be capable of being connected to an identifier oligonucleotide containing codons informative of the identity of the chemical entities which have participated in the formation of the encoded molecule. In a certain embodiment, primers are designed complementary to the codons appearing on the identifier oligonucleotide, and the presence, absence or relative abundance of a codon is evaluated by mixing a primer with the identifier oligonucleotide in the presence of a polymerase and substrate (deoxy)ribonucleotide triphosphates measuring the extension reaction. In another aspect, the invention provides a method for selecting compounds which binds to a target.

Abstract: Disclosed herein include systems, methods, compositions, and kits for associating single cell sequencing data with phenotypic data and/or agent exposure. In some embodiments, cell indexing oligonucleotides comprising cell indexing sequences are associated with cells. In some embodiments, synthetic particles are associated with stochastic labels comprising cell indexing sequences. Sequential hybridizing and imaging of probes annealing to a cell indexing sequence can generate a spatial index. The spatial index can be employed to identify of the partition from which a sequenced nucleic acid target molecule originated.

Abstract: Methods of double-stranded nucleic acid sequence determination and assembly that are able to identify insertions, deletions, repeat region sizes and genomic rearrangements, for example, are disclosed herein, which can use relatively large labeled nucleic acid fragments to analyze the structure of even larger genetic regions. In some embodiments these methods involve the use of certain parameters which unexpectedly improve overall method performance. In some embodiments these methods involve sample labeling that does not result in the formation of single-stranded nucleic acid fragment labeling intermediaries.