SINGLE CELL ANALYSIS OF TRANSPOSASE ACCESSIBLE CHROMATIN
First Claim
1. A method of generating barcoded nucleic acid fragments, comprising:
- (a) generating a plurality of partitions, wherein at least a subset of said plurality of partitions comprises;
(i) a single biological particle from a plurality of biological particles, wherein said single biological particle comprises template DNA molecules and template RNA molecules;
(ii) a plurality of first barcode oligonucleotide molecules comprising a barcode sequence;
(iii) a plurality of transposon end oligonucleotide molecules comprising a transposon end sequence;
(iv) a plurality of transposase molecules;
(v) a plurality of second barcode oligonucleotide molecules comprising a barcode sequence and a capture sequence; and
(vi) a plurality of reverse transcriptase molecules;
(b) generating a plurality of template DNA fragments by subjecting said subset of said plurality of partitions to conditions sufficient to cause transposition of said transposon end oligonucleotide molecules into said template DNA with the aid of a transposase-nucleic acid complex comprising a transposase molecule from said plurality of transposase molecules and a transposon end oligonucleotide molecule from said plurality of transposon end oligonucleotide molecules;
(c) generating a barcoded DNA fragment using a barcode oligonucleotide molecule from said plurality of first barcode oligonucleotide molecules and a template DNA fragment from said plurality of template DNA fragments; and
(d) generating a barcoded cDNA molecule from said template RNA molecules by reverse transcription using a barcode oligonucleotide molecule from said plurality of second barcode oligonucleotide molecules.
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Abstract
Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.
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Citations
30 Claims
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1. A method of generating barcoded nucleic acid fragments, comprising:
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(a) generating a plurality of partitions, wherein at least a subset of said plurality of partitions comprises;
(i) a single biological particle from a plurality of biological particles, wherein said single biological particle comprises template DNA molecules and template RNA molecules;
(ii) a plurality of first barcode oligonucleotide molecules comprising a barcode sequence;
(iii) a plurality of transposon end oligonucleotide molecules comprising a transposon end sequence;
(iv) a plurality of transposase molecules;
(v) a plurality of second barcode oligonucleotide molecules comprising a barcode sequence and a capture sequence; and
(vi) a plurality of reverse transcriptase molecules;(b) generating a plurality of template DNA fragments by subjecting said subset of said plurality of partitions to conditions sufficient to cause transposition of said transposon end oligonucleotide molecules into said template DNA with the aid of a transposase-nucleic acid complex comprising a transposase molecule from said plurality of transposase molecules and a transposon end oligonucleotide molecule from said plurality of transposon end oligonucleotide molecules; (c) generating a barcoded DNA fragment using a barcode oligonucleotide molecule from said plurality of first barcode oligonucleotide molecules and a template DNA fragment from said plurality of template DNA fragments; and (d) generating a barcoded cDNA molecule from said template RNA molecules by reverse transcription using a barcode oligonucleotide molecule from said plurality of second barcode oligonucleotide molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30)
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28. The method of claim 28, further comprising sequencing said barcoded cDNA molecule, or a derivative thereof.
Specification