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Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations

Inactive Publication Date: 2015-12-31
10X GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for extracting RNA from individual cells, which involves releasing the nucleic acids from the cells and subjecting them to reverse transcription using oligonucleotides. The reverse transcription can occur in a discrete partition and can result in the formation of one or more nucleic acid sequences. The process can also involve the use of a random priming sequence or a poly-T sequence to facilitate the hybridization of the oligonucleotides to the nucleic acids. The technical effect of this patent is the improved accuracy and efficiency of extracting high-quality RNA from individual cells.

Problems solved by technology

Despite these advances in biological characterization, many challenges still remain unaddressed, or relatively poorly addressed by the solutions currently being offered.

Method used

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  • Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations
  • Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations
  • Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations

Examples

Experimental program
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example i

Cellular RNA Analysis Using Emulsions

[0159]In an example, reverse transcription with template switching and cDNA amplification (via PCR) is performed in emulsion droplets with operations as shown in FIG. 9A. The reaction mixture that is partitioned for reverse transcription and cDNA amplification (via PCR) includes 1,000 cells or 10,000 cells or 10 ng of RNA, beads bearing barcoded oligonucleotides / 0.2% Tx-100 / 5× Kapa buffer, 2× Kapa HS HiFi Ready Mix, 4 μM switch oligo, and Smartscribe. Where cells are present, the mixture is partitioned such that a majority or all of the droplets comprise a single cell and single bead. The cells are lysed while the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of mRNA that is released from the cell as in operation 950. The poly-T segment is extended in a reverse transcription reaction as in operation 952 and the cDNA transcript is amplified as in operation...

example ii

Cellular RNA Analysis Using Emulsions

[0161]In another example, reverse transcription with template switching and cDNA amplification (via PCR) is performed in emulsion droplets with operations as shown in FIG. 9A. The reaction mixture that is partitioned for reverse transcription and cDNA amplification (via PCR) includes Jurkat cells, beads bearing barcoded oligonucleotides / 0.2% TritonX-100 / 5× Kapa buffer, 2× Kapa HS HiFi Ready Mix, 4 μM switch oligo, and Smartscribe. The mixture is partitioned such that a majority or all of the droplets comprise a single cell and single bead. The cells are lysed while the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of mRNA that is released from the cell as in operation 950. The poly-T segment is extended in a reverse transcription reaction as in operation 952 and the cDNA transcript is amplified as in operation 954. The thermal cycling conditions are 42° C...

example iii

RNA Analysis Using Emulsions

[0162]In another example, reverse transcription is performed in emulsion droplets and cDNA amplification is performed in bulk in a manner similar to that as shown in FIG. 9C. The reaction mixture that is partitioned for reverse transcription includes beads bearing barcoded oligonucleotides, 10 ng Jurkat RNA (e.g., Jurkat mRNA), 5× First-Strand buffer, and Smartscribe. The barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of the RNA as in operation 961. The poly-T segment is extended in a reverse transcription reaction as in operation 963. The thermal cycling conditions for reverse transcription are one cycle at 42° C. for 2 hours and one cycle at 70° C. for 10 min. Following thermal cycling, the emulsion is broken and RNA and cDNA transcripts are denatured as in operation 962. A second strand is then synthesized by primer extension with a primer having a biotin tag a...

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Abstract

Methods, compositions and systems for analyzing individual cells or cell populations through the partitioned analysis of contents of individual cells or cell populations. Individual cells or cell populations are co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the contents of a given cell or cell population, and subsequently analyzing the cell's contents and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of the cell's nucleic acids through sequencing.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Patent Application No. 62 / 017,558 filed Jun. 26, 2014 and U.S. Provisional Patent Application No. 62 / 061,567 filed Oct. 8, 2014 each of which applications is herein incorporated by reference in its entirety for all purposes.BACKGROUND[0002]Significant advances in analyzing and characterizing biological and biochemical materials and systems have led to unprecedented advances in understanding the mechanisms of life, health, disease and treatment. Among these advances, technologies that target and characterize the genomic make up of biological systems have yielded some of the most groundbreaking results, including advances in the use and exploitation of genetic amplification technologies, and nucleic acid sequencing technologies.[0003]Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical contexts, including diagnostics, prognostics, biotechnology, and fo...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1065C40B20/04C40B50/16C12Q1/6806C12Q2525/161C12Q2535/122C12Q2545/114C12Q2563/149C12Q2563/159C12Q2563/179C12Q1/6816C12Q1/6874C12Q2565/629C12Q1/6804C12Q1/683C12Q2525/191C12Q2537/143C12Q2537/149
Inventor HINDSON, BENJAMINHINDSON, CHRISTOPHERSCHNALL-LEVIN, MICHAELNESS, KEVINJAROSZ, MIRNASAXONOV, SERGEHARDENBOL, PAULBHARADWAJ, RAJIVZHENG, GRACEBELGRADER, PHILLIP
Owner 10X GENOMICS
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