Systems and methods for processing RNA from cells
Inventors
Alvarado Martinez, Luigi Jhon • Ramachandran Iyer, Eswar Prasad • Bell, Jason
Assignees
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Publication Number
US-12606860-B2
Publication Date
2026-04-21
Expiration Date
Abstract
Provided herein are methods, compositions, and systems for processing nucleic acids from individual cells or cell populations. Cells are co-partitioned and processed, allowing for analysis of a variety of types of RNA from cells while minimizing ribosomal RNA species. The present invention enables high-throughput measurement of full length RNA from single cells with a streamlined workflow.
Core Innovation
The invention provides a method for assaying ribonucleic acid (RNA) molecules that includes depleting ribosomal RNA (rRNA) molecules from RNA molecules in a cell or nucleus, yielding a plurality of remaining RNA molecules. The method further fragments the remaining RNA molecules in the cell or nucleus to yield a plurality of RNA fragments.
The method provides a plurality of nucleic acid barcode molecules that comprise a common barcode sequence. An RNA fragment and a nucleic acid barcode molecule having that common barcode sequence are used to generate a barcoded nucleic acid molecule.
The described approach supports high-throughput full-length or extended RNA measurement in the context of individual cells or cell populations by using co-partitioning. RNA processing is combined with nucleic-acid barcoding, including the generation of barcoded cDNA using hybridization/extension or covalent attachment/ligation, followed by partitioning with nucleic-acid barcode molecules delivered through droplets or wells.
Claims Coverage
The claim set centers on assaying RNA by depleting rRNA, fragmenting remaining RNA in a cell or nucleus, and generating barcoded nucleic acid molecules using nucleic acid barcode molecules that share a common barcode sequence. Dependent claims refine this workflow with TGIRT reverse transcription, choices for chemical versus enzymatic fragmentation, specified non-poly-adenylated RNA categories, and nucleic acid reactions using a T4KQ ligase.
In-cell or in-nucleus rRNA depletion and fragmentation for RNA fragments
Depleting ribosomal RNA (rRNA) molecules from RNA molecules in said cell or nucleus to yield remaining RNA molecules; fragmenting, in said cell or nucleus, the remaining RNA molecules to yield RNA fragments.
Nucleic acid barcodes with a common barcode sequence
Providing a plurality of nucleic acid barcode molecules wherein nucleic acid barcode molecules comprise a common barcode sequence.
Generating barcoded nucleic acid from RNA fragments and common-barcode molecules
Using an RNA fragment of the plurality of RNA fragments and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded nucleic acid molecule.
TGIRT reverse-transcription with an RNA molecule hybridized to the barcode
Nucleic acid reaction using a thermostable group II intron reverse transcriptase (TGIRT), where the nucleic acid barcode molecule has an RNA molecule hybridized to it.
Chemical or enzymatic fragmentation choice
Fragmenting the plurality of remaining RNA molecules by performing the fragmenting step as either a chemical fragmentation process or an enzymatic fragmentation process.
Chemical fragmentation modalities using divalent metal cations/heating or reactive oxygen species
Limiting the chemical fragmentation process to use divalent metal cations and heating, or to use a reactive oxygen species.
Non-poly-adenylated RNA categories as starting RNA molecules
Using non-poly-adenylated RNA molecules selected from miRNA, long non-coding RNA, short non-coding RNA, tRNA, RNA isoforms, snoRNA, small RNA, piRNA, or circular RNA.
Nucleic acid reaction using a T4KQ ligase
Performing a nucleic acid reaction using a T4KQ ligase.
The core workflow is rRNA depletion and in-cell or in-nucleus fragmentation to produce RNA fragments, followed by generating barcoded nucleic acid molecules using nucleic acid barcode molecules with a common barcode sequence. The dependent claims further specify barcode and reaction components, fragmentation options, RNA starting-material classes, and a ligase component.
Stated Advantages
Enables high-throughput full-length/extended RNA measurement.
Documented Applications
Assaying RNA molecules from a cell or nucleus comprising RNA molecules, including in the context of individual cells or cell populations using co-partitioning (droplets/wells).
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