# US 2020/0399686 A1 · Method and apparatus for simultaneous targeted sequencing of dna, rna and protein > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399686 A1 - Document ID: US20200399686A1 - Status: Published application - Kind: A1 - Application number: 16/882,289 - Filed: 2020-05-22 - Published: 2020-12-24 - Earliest claimed: 2019-05-22 - Assignees: Mission Bio, Inc. - Applicants: Mission Bio, Inc. - Inventors: Dalia Dhingra; Aik Ooi; Pedro Mendez; David Ruff - Web page: https://patentgrep.com/patent/US20200399686A1 ## CPC - C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase - C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass} - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q 2521/107: RNA dependent DNA polymerase,(i.e. reverse transcriptase) - C12Q 2521/537: Protease - 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 - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Provided herein are methods and systems for the simultaneous targeted detection and sequencing of DNA, RNA, and Protein. In typical embodiments, the DNA, RNA, and Proteins are detected, characterized, and sequenced using just a single mammalian cell. One embodiment of detecting and characterizing DNA, RNA, or protein from a mammalian cell includes encapsulating a single cell in a drop and performing a protease digest on the encapsulated cell drop, performing a reverse transcriptase reaction; performing a droplet merger with barcoding PCR reagents and barcoding beads; performing a PCR reaction to attach the cell barcodes to the DNA targeted amplicons, RNA targeted amplicons, and protein tag amplicons, where all amplicons from the same emulsion contain the same cell barcode; and detecting and characterizing a DNA, RNA, or protein amplicon by sequencing the cell barcode incorporated into each amplicon. ## Claims ### Claim 1 A multiomic detection and characterization method for detecting DNA, RNA, or protein from a cell, comprising encapsulating a cell in a drop comprising a reaction mixture comprising a protease; performing a protease digest on the encapsulated cell drop with the protease to produce a cell lysate; providing a reverse transcriptase and performing a reverse transcription reaction; performing a droplet merger with barcoding PCR reagents and barcoding beads; performing a PCR reaction to attach the cell barcodes to the DNA targeted amplicons, RNA targeted amplicons, and protein tag amplicons, wherein all amplicons from the same emulsion contain the same cell barcode; and detecting and characterizing a DNA, RNA, or protein amplicon by sequencing the cell barcode incorporated into each amplicon. ### Claim 2 (depends on 1) A method according to claim 1, wherein DNA, RNA, and proteins are detected and characterized ### Claim 3 (depends on 1) A method according to claim 1, wherein DNA, RNA, and proteins are detected and characterized from a single cell. ### Claim 4 (depends on 1) A method according to claim 1, wherein the reverse transcription reaction is performed in the same drop as the protease digest. ### Claim 5 (depends on 1) A method according to claim 1, further comprising performing a capture of DNA and RNA amplicons to a solid phase, wherein protein tag amplicons are separated from DNA and RNA amplicons. ### Claim 6 (depends on 1) A method according to claim 1, wherein a droplet merger with the cell lysate and reverse transcription is performed before the PCR reaction is performed. ### Claim 7 (depends on 1) A method according to claim 1, wherein the protease and reverse transcriptase reactions are performed on a PCR thermocycler and later transferred to another instrument for processing and analysis. ### Claim 8 (depends on 4) A method according to claim 4, wherein the instrument for processing and analysis comprises a Mission Bio, Tapestri™ system and the droplet merger with barcoding PCR reagents and barcoding beads is performed on this instrument. ### Claim 9 (depends on 1) A method according to claim 1, further comprising performing a nucleic acid concentration step. ### Claim 10 (depends on 1) A method according to claim 1, further comprising making a protein library. ### Claim 11 (depends on 1) A method according to claim 1, further comprising making a DNA library. ### Claim 12 (depends on 1) A method according to claim 1, further comprising making a RNA library. ### Claim 13 (depends on 1) A method according to claim 1, further comprising making a DNA, protein, and RNA library. ### Claim 14 (depends on 1) A method according to claim 1, wherein a reverse transcription reaction to produce a reverse transcription product is performed before a nucleic acid amplification step. ### Claim 15 (depends on 1) A method according to claim 1, wherein in the step, of performing a capture of DNA and RNA amplicons to a solid phase, the solid phase is a bead. ### Claim 16 (depends on 1) The method of claim 1, wherein one or more DNA, RNA, and protein listed in FIG. 9 is detected and characterized. ### Claim 17 (depends on 1) The method of claim 1, wherein two or more DNA, RNA, and protein listed in FIG. 9 are detected and characterized. ### Claim 18 (depends on 1) The method of claim 1, wherein three or more DNA, RNA, and protein listed in FIG. 9 are detected and characterized. ### Claim 19 (depends on 1) The method of claim 1, wherein four or more DNA, RNA, and protein listed in FIG. 9 are detected and characterized. ### Claim 20 (depends on 1) The method of claim 1, wherein five or more DNA, RNA, and protein listed in FIG. 9 are detected and characterized.