RNA printing and sequencing devices, methods, and systems

Inventors

Sims, Peter A. • Bose, Sayantan • YUAN, Jinzhou

Assignees

Columbia University

Abstract

Many important biological questions demand single-cell transcriptomics on a large scale. Hence, new tools are urgently needed for efficient, inexpensive manipulation of RNA from individual cells. Described herein are devices, systems, and methods for trapping single-cell lysates in sealed, microwells capable of printing RNA on glass or capturing RNA on beads.

Core Innovation

The invention relates to visualizing and identifying mRNA capture beads in a device comprising a plurality of chambers, microchambers, or microwells, where each chamber contains at least one mRNA capture bead. The mRNA capture beads are labeled with a primer sequence that comprises a unique combination of the presence or absence of members of a set of barcode sequences. Each barcode sequence is unique with respect to the others, and each primer sequence is designed so that optically labelled oligonucleotides hybridize to one and only one correspondingly numbered member of the set of barcode sequences present on the primer sequences.

Optically labelled oligonucleotides are contacted to the plurality of chambers, and unhybridized optically labelled oligonucleotides are removed by washing with a wash buffer. The chambers are then imaged to identify wells that have an mRNA capture bead labeled via hybridization to the first optically labelled oligonucleotide, thereby generating initial imaging data indicating the presence of first barcode sequences. In further embodiments, denaturing and successively repeated hybridization with additional optically labelled oligonucleotides are performed to generate final imaging data indicating a barcode presence/absence combination for the primer sequences.

The mRNA capture bead primer sequences can include oligo(dT), an adapter sequence, and a T7 promoter sequence, and may include a sequencing adapter. The primer sequence architecture supports decoding through optically labelled oligonucleotides and imaging so that bead identity and barcode presence/absence patterns can be determined for at least 32 mRNA capture beads across the device.

Claims Coverage

The provided independent claim set contains 1 independent claim, directed to a method of visualizing mRNA capture beads using optically labelled oligonucleotides, washing, and imaging to generate imaging data indicating presence of barcode sequences. The inventive features are further refined by sequential hybridization with additional labelled oligonucleotides, defined primer sequence elements, denaturing, occupancy constraints, and linking imaging data with sequencing data.

The claim coverage centers on generating imaging data for mRNA capture bead primer barcode presence/absence patterns by hybridizing optically labelled oligonucleotides to primer sequences, removing unhybridized labels by washing, and imaging wells to identify which bead barcode components are present. Dependent refinements include producing final barcode presence/absence combinations using denaturing and repeated hybridization, specifying primer architecture elements including oligo(dT), adapter, T7 promoter, adding sequencing adapters, imposing occupancy constraints, and linking final imaging data with sequencing data.

Stated Advantages

Documented Applications