The present disclosure provides methods, compositions and systems for analyzing individual cells or cell populations through a partitioned analysis of contents of individual cells or cell populations, such as cancer cells and cells of the immune system. Individual cells or cell populations may be co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the content of a given cell or cell population, and subsequently analyzing the content of the cell and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of nucleic acid(s) from the cell through sequencing.

Single-cell RNA sequencing (scRNAseq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on the adaptive immune system's T and B cells, scRNAseq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (TCR or BCR, respectively), and link it to the functional state inferred from transcriptome analysis. Computational approaches to infer clonality and maturation status (for BCR only) from scRNAseq datasets of T and B cells have been developed but there are cumbersome and not costly effective. The inventors have now developed a FACS-based 5′-end RNAseq method, in particular a FACS-based 5′-end scRNAseq method, for cost effective integrative analysis of B and T cell transcriptome and paired BCR and TCR repertoire in phenotypically defined B and T cell subsets. In particular, the method of the present invention includes a reverse transcription step that uses a number of different well specific template switching oligonucleotides (TSO) to introduce a well-specific DNA barcode in the 5′-end of cDNAs.

Provided herein are automated apparatus and methods for the identification of microorganisms in various samples. The disclosure solves existing challenges encountered in identifying and distinguishing various types of microorganisms, including viruses and bacteria in a timely, efficient, and automated manner by library preparation and sequencing.

A method for characterizing proteins, including steps of (a) detecting a plurality of proteins, wherein individual proteins of the plurality are associated with unique identifiers, wherein the detecting distinguishes the identities of the individual proteins and the unique identifiers associated with the individual proteins; (b) digesting the proteins to form peptides, wherein the peptides from each protein are associated with the unique identifiers for the respective individual protein; (c) detecting the peptides and associated unique identifiers, wherein the detecting distinguishes characteristics of individual peptides, and wherein the detecting distinguishes unique identifiers associated with the individual peptides; and (d) correlating characteristics detected in step (c) with individual proteins detected in step (a) based on the unique identifiers associated with the individual proteins and the peptides.

In an aspect, the present disclosure provides a method for determining a methylation status comprises: providing a biological sample of nucleic acid molecules; partitioning at least a subset of the nucleic acid molecules in the biological sample based on the methylation status of the nucleic acid molecules into a plurality of partitioned sets; digesting at least a subset of the one or more partitioned sets in the plurality of partitioned sets with at least one methylation sensitive restriction enzyme; enriching at least a subset of the nucleic acid molecules in the plurality of partitioned sets for genomic regions of interest, wherein the at least a subset of the nucleic acid molecules comprises digested nucleic acid molecules in the one or more partitioned sets; and determining methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioned sets.

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.

The present disclosure relates to methods of identifying RNA targets of RNA binding proteins. In aspects, the disclosure relates to a method of identifying RNA molecules bound by RNA binding proteins. Some embodiments of the present disclosure relate to a method that can definitively identify direct RNA-target interactions with targeted proteins without the requirement for immunoprecipitation or gel extraction. In some embodiments, the method may include combining multiple antibodies in the same sample.

The present disclosure relates to methods of identifying RNA targets of RNA binding proteins. In aspects, the disclosure relates to a method of identifying RNA molecules bound by RNA binding proteins. Some embodiments of the present disclosure relate to a method that can definitively identify direct RNA-target interactions with targeted proteins without the requirement for immunoprecipitation or gel extraction. In some embodiments, the method may include combining multiple antibodies in the same sample.

Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.

Aspects of the present disclosure include methods of producing modified polypeptides and modified polypeptide-ribosome or polypeptide-mRNA complexes, and methods of screening polynucleotide and polypeptide libraries. The present disclosure also provides polypeptide libraries useful in screening for single molecule phenotypes. Also provided are kits useful for producing polypeptides capable of being modified using methods disclosed herein.