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US 2022/0033893 A1Published application
Single Cell Genomic Sequencing Using Hydrogel Based Droplets
AssigneeThe Regents of the University of California
InventorsFreeman Lan, Benjamin Demaree, Iain Clark, Adam R. Abate
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Abstract
The present disclosure provides ultrahigh-throughput single cell genomic sequencing methods, referred to herein as “SiC-seq”, which methods include encapsulating single cells in molten gel droplets to facilitate bulk cell lysis and purification of genomic DNA in microgels. Systems and devices for practicing the subject methods are also provided.
Claims37
A method of sequencing single cell genomic DNA, the method comprising:
- encapsulating a population of single cells in molten gel droplets to provide a population of molten gel droplets, wherein each molten gel droplet of the population contains zero or one cell;
- solidifying the population of molten gel droplets to provide a population of solidified microgel droplets;
- breaking the emulsions of the solidified microgel droplets to provide a population of solidified microgels;
- exposing the population of solidified microgels in bulk to lysis conditions sufficient to lyse cells contained within the population of solidified microgels;
- purifying genomic DNA from cells contained within the population of solidified microgels in bulk to provide a population of solidified microgels comprising purified genomic DNA;
- encapsulating the population of solidified microgels comprising purified genomic DNA into droplets to provide a population of purified genomic DNA-containing droplets;
- fragmenting the purified genomic DNA within the population of purified genomic DNA-containing droplets to provide a population of fragmented genomic DNA-containing droplets;
- barcoding the fragmented genomic DNA or an amplification product thereof in the population of fragmented genomic DNA-containing droplets to provide a population of barcoded, fragmented genomic DNA-containing droplets;
- purifying barcoded, fragmented genomic DNA from the barcoded, fragmented genomic DNA-containing droplets to provide purified, barcoded, fragmented genomic DNA;
- and
- sequencing the purified, barcoded, fragmented genomic DNA.
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Dependencies are inferred from the claim text.