Methods for analyzing nucleic acids from single cells

  • US 10,280,459 B1
  • Filed: 01/29/2019
  • Issued: 05/07/2019
  • Est. Priority Date: 08/20/2009
  • Status: Active Grant
First Claim
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1. A method for multiplexed analysis of nucleic acids from single cells, the method comprising:

  • (a) providing a sample comprising a plurality of cells, wherein a single cell of the plurality of cells comprises a plurality of sample polynucleotides;

    (b) performing primer extension to generate a plurality of tagged polynucleotides from said plurality of sample polynucleotides and a plurality of oligonucleotide tags, wherein a tagged polynucleotide of the plurality of tagged polynucleotides comprises;

    (i) a sample sequence from a sample polynucleotide of the plurality of sample polynucleotides;

    (ii) a first tag sequence distinguishing said sample polynucleotide from sample polynucleotides originating from other single cells; and

    (iii) a second tag sequence distinguishing said sample polynucleotide from other sample polynucleotides originating from said same single cell;

    (c) amplifying said tagged polynucleotide, thereby generating a plurality of amplified polynucleotides; and

    (d) sequencing said plurality of amplified polynucleotides to determine sequences of the amplified polynucleotides corresponding to the sample sequence, the first tag sequence, and the second tag sequence of the tagged polynucleotide; and

    (e) using the sequences determined in step (d) to count sample polynucleotides for multiple different sample polynucleotides of multiple different single cells of said plurality of cells.

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