# US 12,110,549 B2 · Methods and systems for processing polynucleotides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,110,549 B2 - Document ID: US12110549B2 - Status: Granted - Kind: B2 - Application number: 17/822,117 - Filed: 2022-08-24 - Published: 2023-06-29 - Granted: 2024-10-08 - Earliest claimed: 2016-12-22 - Assignees: 10x Genomics, Inc. - Applicants: 10x Genomics, Inc. - Inventors: Phillip Belgrader; Zachary Bent; Rajiv Bharadwaj; Vijay Kumar Sreenivasa Gopalan; Josephine Harada; Christopher Hindson; Mohammad Rahimi Lenji; Michael Ybarra Lucero; Geoffrey McDermott; Elliott Meer; Tarjei Sigurd Mikkelsen; Christopher Joachim O'Keeffe; Katherine Pfeiffer; Andrew D. Price; Paul Ryvkin; Serge Saxonov; John R. Stuelpnagel; Jessica Michele Terry; Tobias Daniel Wheeler; Indira Wu; Solongo Batjargal Ziraldo; Stephane Claude Boutet; Sarah Taylor; Niranjan Srinivas - Web page: https://patentgrep.com/patent/US12110549B2 ## CPC - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] - C12Q 1/6869: Methods for sequencing - C12Q 2525/191: incorporating an adaptor - C12Q 2537/143: Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis - C12Q 2563/179: the label being a nucleic acid - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization. ## Claims ### Claim 1 A method, comprising: (a) providing a partition comprising: (i) a labeled cell or a labeled organelle, wherein the labeled cell or the labeled organelle comprises a surface labeling agent, wherein the surface labeling agent is a small molecule attached to a reporter nucleic acid barcode molecule comprising a reporter barcode sequence, wherein the small molecule comprises a molecular weight of no greater than 1,000 Daltons (Da); and (ii) a partition nucleic acid barcode molecule comprising a partition barcode sequence; and (b) using the reporter nucleic acid barcode molecule and the partition nucleic acid barcode molecule to generate a barcoded nucleic acid molecule comprising (1) the reporter barcode sequence or a reverse complement thereof and (2) the partition barcode sequence or a reverse complement thereof. ### Claim 2 (depends on 1) The method of claim 1, wherein the surface labeling agent is directly linked to the reporter nucleic acid barcode molecule. ### Claim 3 (depends on 1) The method of claim 1, wherein the surface labeling agent is indirectly linked to the reporter nucleic acid barcode molecule. ### Claim 4 (depends on 1) The method of claim 1, further comprising: (c) sequencing the barcoded nucleic acid molecule or a derivative or an amplification product thereof to identify (1) the reporter barcode sequence, and (2) the partition barcode sequence. ### Claim 5 (depends on 1) The method of claim 1, wherein the labeled cell or the labeled organelle further comprises a messenger ribonucleic nucleic acid (mRNA) molecule comprising a mRNA nucleic acid sequence, and wherein: the partition of (a) further comprises a second partition nucleic acid barcode molecule comprising the partition barcode sequence; and (b) further comprises using the mRNA molecule and the second partition nucleic acid barcode molecule to generate a second barcoded nucleic acid molecule comprising (1) the mRNA nucleic acid sequence or a reverse complement thereof and (2) the partition barcode sequence or a reverse complement thereof. ### Claim 6 (depends on 5) The method of claim 5, further comprising: (c) sequencing the second barcoded nucleic acid molecule or a derivative or an amplification product thereof. ### Claim 7 (depends on 1) The method of claim 1, wherein the labeled cell or the labeled organelle further comprises a second labeling agent comprising (1) an antibody or binding fragment thereof and (2) a second reporter nucleic acid barcode molecule comprising a second reporter barcode sequence, and wherein: the partition of (a) further comprises a second partition nucleic acid barcode molecule comprising the partition barcode sequence; and (b) further comprises using the second reporter nucleic acid barcode molecule and the second partition nucleic acid barcode molecule to generate a second barcoded nucleic acid molecule comprising (1) the second reporter barcode sequence or a reverse complement thereof and (2) the partition barcode sequence or a reverse complement thereof. ### Claim 8 (depends on 7) The method of claim 7, further comprising: (c) sequencing the second barcoded nucleic acid molecule or a derivative or an amplification product thereof. ### Claim 9 (depends on 7) The method of claim 7, wherein the antibody or binding fragment thereof is directly attached to the second reporter nucleic acid barcode molecule. ### Claim 10 (depends on 7) The method of claim 7, wherein the antibody or binding fragment thereof is indirectly attached to the second reporter nucleic acid barcode molecule. ### Claim 11 (depends on 1) The method of claim 1, wherein the barcoded nucleic acid molecule is generated via one or more extension reactions. ### Claim 12 (depends on 1) The method of claim 1, wherein the partition nucleic acid barcode molecule is coupled to a support. ### Claim 13 (depends on 12) The method of claim 12, wherein the partition nucleic acid barcode molecule is releasably coupled to the support. ### Claim 14 (depends on 12) The method of claim 12, wherein the support is a bead. ### Claim 15 (depends on 14) The method of claim 14, wherein the bead is a solid bead. ### Claim 16 (depends on 15) The method of claim 15, wherein the solid bead is a magnetic bead. ### Claim 17 (depends on 1) The method of claim 1, wherein the partition is a well. ### Claim 18 (depends on 1) The method of claim 1, wherein the partition is a droplet in an emulsion. ### Claim 19 (depends on 1) The method of claim 1, wherein the surface labeling agent is attached to a surface feature of the labeled cell or the labeled organelle. ### Claim 20 (depends on 1) The method of claim 1, wherein the method further comprises, before (b), lysing the labeled cell or the labeled organelle. ### Claim 21 (depends on 1) The method of claim 1, wherein the small molecule is selected from the group consisting of cholesterol, tocopherol, lignoceric acid, palmitic acid, and derivatives thereof. ### Claim 22 (depends on 1) The method of claim 1, wherein the small molecule is an organic compound. ### Claim 23 (depends on 1) The method of claim 1, wherein the surface labelling agent is linked to the reporter nucleic acid barcode molecule via a click chemistry linker. ### Claim 24 (depends on 1) The method of claim 1, wherein the partition nucleic acid barcode molecule comprises a unique molecular identification (UMI) sequence. ### Claim 25 (depends on 19) The method of claim 19, wherein the surface feature is a surface protein. ### Claim 26 A method of analyzing a cell or an organelle, comprising: (a) partitioning a labeled cell or a labeled organelle and a bead comprising a plurality of partition nucleic acid barcode molecules into a partition, wherein: (i) the labeled cell or the labeled organelle comprises (1) a surface labeling agent that is a small molecule linked to a reporter nucleic acid barcode molecule comprising a reporter nucleic acid barcode sequence, and (2) a messenger ribonucleic acid (mRNA) analyte comprising an mRNA sequence, wherein the small molecule comprises a molecular weight of no greater than 1,000 Daltons (Da), and (ii) the plurality of partition nucleic acid barcode molecules comprises (1) a first partition nucleic acid barcode molecule comprising a partition barcode sequence, a first unique molecular identifier (UMI) sequence, and a poly-T sequence and (2) a second partition nucleic acid barcode molecule comprising the partition barcode sequence, a second unique molecular identifier (UMI) sequence, and the poly-T sequence; (b) hybridizing the poly-T sequence of the first partition nucleic acid barcode molecule to the reporter nucleic acid barcode molecule and extending the first partition nucleic acid barcode molecule using the reporter nucleic acid barcode molecule as a template to generate a first barcoded nucleic acid molecule comprising (1) the partition barcode sequence, (2) the first UMI sequence, and (3) a reverse complement of the reporter nucleic acid barcode sequence; (c) hybridizing the poly-T sequence of the second partition nucleic acid barcode molecule to the mRNA analyte and extending the second partition nucleic acid barcode molecule using the mRNA analyte as a template to generate a second barcoded nucleic acid molecule comprising (1) the partition barcode sequence, (2) the second UMI sequence, and (3) a reverse complement of the mRNA sequence; and (d) sequencing (i) the first barcoded nucleic acid molecule or a derivative or an amplification product thereof, and (ii) the second barcoded nucleic acid molecule or a derivative or an amplification product thereof. ### Claim 27 (depends on 26) The method of claim 26, wherein the partition is a droplet in an emulsion or a well. ### Claim 28 (depends on 26) The method of claim 26, wherein the hybridizing of (b) and (c) occurs in the partition. ### Claim 29 (depends on 26) The method of claim 26, wherein the hybridizing and the extending of (b) and (c) occurs in the partition.