Methods for generating barcoded nucleic acid molecules using fixed cells
Assignee10x Genomics, Inc.
InventorsJoshua Delaney, Shalini Gohil, Jill Herschleb, Adam Lowe, Albert Kim, Meiliana Tjandra
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Abstract
The present disclosure provides compositions and methods for using fixed biological samples in partition-based assays. In at least one embodiment, the disclosure provides a composition comprising a fixed biological sample and an un-fixing agent contained in a partition, such as a discrete droplet. In some embodiments, the disclosure provides un-fixing agent compounds capable of catalytically cleaving crosslinks in fixed biological samples, particularly crosslinked nucleic acids, such as RNA.
Claims15
A method for generating a plurality of barcoded nucleic acid molecules using fixed cells, the method comprising:
- (a) providing a plurality of fixed cells, wherein a fixed cell of said plurality of fixed cells comprises a plurality of crosslinked nucleic acid molecules;
- (b) un-fixing said fixed cell with an un-fixing agent to provide an un-fixed cell comprising a plurality of un-crosslinked nucleic acid molecules from said plurality of crosslinked nucleic acid molecules;
- and
- (c) generating the plurality of barcoded nucleic acid molecules from said plurality of un-crosslinked nucleic acid molecules, wherein a barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises i) a sequence corresponding to an un-crosslinked nucleic acid molecule of said plurality of said un-crosslinked nucleic acid molecules or a complement thereof, and ii) a barcode sequence or a complement thereof, wherein said un-fixing agent comprises a compound selected from the group consisting of (4-aminopyridin-3-yl)phosphonic acid, (3-aminopyridin-2-yl)phosphonic acid, (5-aminopyrimidin-4-yl)phosphonic acid, and a combination thereof.
Dependencies are inferred from the claim text.