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US 2024/0182964 A1Published application
Method for preparation and high-throughput microbial single-cell rna sequencing of bacteria
AssigneeUniversity of Washington
InventorsGeorg Seelig, Anna Kuchina, Leandra Brettner, William DePaolo
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Abstract
Methods and kits for uniquely labeling nucleic acid molecules within a plurality of microbial cells are described. In an embodiment, the method comprises fixing and permeabilizing the plurality of microbial cells; dissociating microbial cell aggregates within a suspension comprising the plurality of microbial cells; reverse transcribing mRNA within the plurality of microbial cells to provide cDNA; and combinatorially labelling the cDNA to provide labelled cDNA.
Claims30
A kit for labelling nucleic acids within a microbial cell, the kit comprising:
- (a) a reverse transcriptase enzyme;
- (b) a cell wall-degradation or a permeabilization enzyme configured to degrade microbial cell walls;
- (c) at least one reverse transcription (RT) primer comprising a 5′ overhang sequence;
- and
- (d) a first set of nucleic acid tags, wherein each of the first set of nucleic acid tags comprises:
- (i) a first strand comprising:
- a primary labeling sequence comprising a 3′ and a 5′ end, wherein multiple distinct primary labeling sequences are present among the nucleic acid tags of the first set;
- a primary 3′ hybridization sequence extending from the 3′ end of the primary labeling sequence;
- and
- a primary 5′ hybridization sequence extending from the 5′ end of the primary labeling sequence;
- and
- (ii) a second strand comprising:
- a first portion complementary to the 5′ overhang sequence of the reverse transcription primer, and
- a second portion complementary to the primary 3′ hybridization sequence.
11 more elements
Dependencies are inferred from the claim text.