In situ combinatorial labeling of cellular molecules
Inventors
Seelig, Georg • Rosenberg, Alexander B. • ROCO, Charles
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12234501-B2
Publication Date
2025-02-25
Expiration Date
Abstract
Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.
Core Innovation
The invention provides a method of labeling RNA molecules with nucleus-specific tags by fixing and permeabilizing a plurality of nuclei, wherein each of the plurality of fixed, permeabilized nuclei comprises RNA, and wherein the plurality of nuclei is fixed and permeabilized at a temperature below about 8°C. The nuclei are handled through sequential combining and dividing steps, including dividing into a first plurality of aliquots and later dividing combined nuclei into a second plurality of aliquots and then into a plurality of samples.
RNA molecules within nuclei of the first plurality of aliquots are reverse transcribed to generate complementary DNA (cDNA) molecules using RT primers that include a poly(T) sequence or a random nucleotide sequence, an RT primer barcode sequence, and a 5′ overhang comprising a 5′ overhang sequence located 5′ of the poly(T) or the random nucleotide sequence. The 5′ overhang sequence is the same in all RT primers used in the first plurality of aliquots, and following reverse transcription the 5′ overhang sequence is present at the 5′ end of each cDNA molecule, while multiple distinct RT primer barcode sequences are present and are specific to individual aliquots.
Tagged cDNA molecules are then generated by coupling nucleic acid tags to the cDNA molecules within nuclei of the second plurality of aliquots, where each nucleic acid tag comprises a tag barcode sequence and a 3′ hybridization sequence located 3′ of the barcode sequence and/or a 5′ hybridization sequence located 5′ of the barcode sequence. Multiple distinct tag barcode sequences are present among the nucleic acid tags used in the second plurality of aliquots and are specific to individual aliquots, after which the nuclei are lysed to release the tagged cDNA molecules into sample lysates.
Claims Coverage
The only independent claim provided covers a nucleus-specific split-pool labeling workflow that uses aliquot-specific RT primer barcode sequences in fixed and permeabilized nuclei at below about 8°C, followed by aliquot-specific nucleic acid tag barcode coupling, and final lysis to release tagged cDNA in sample lysates. The claim includes at least nine major inventive stages: fixation and permeabilization, first aliquoting, barcode-associated RT with a shared 5′ overhang, combining, second aliquoting, tag coupling with barcode plus hybridization sequences, combining, sample partitioning, and lysis to release tagged cDNA.
Temperature-limited fixation and permeabilization of nuclei for RNA labeling
Fixing and permeabilizing a plurality of nuclei comprising RNA at a temperature below about 8°C.
Aliquot-specific RT primer barcoding during in-nucleus reverse transcription
Dividing nuclei into first aliquots and reverse transcribing RNA to generate cDNA using RT primers that include an RT primer barcode sequence, where multiple distinct RT primer barcode sequences are present and are specific to individual aliquots, together with a poly(T) sequence or random nucleotide sequence and a 5′ overhang.
Shared 5′ overhang positioned at the cDNA 5′ end
Using RT primers with a 5′ overhang comprising a 5′ overhang sequence that is the same in all RT primers used in the first plurality of aliquots, wherein following reverse transcription the 5′ overhang sequence is present at the 5′ end of each cDNA molecule.
Second-stage nucleic acid tag barcoding with hybridization sequences
Dividing combined nuclei into second aliquots and coupling nucleic acid tags to the cDNA molecules to generate tagged cDNA molecules, where each nucleic acid tag comprises a tag barcode sequence and a 3′ hybridization sequence and/or a 5′ hybridization sequence, and where multiple distinct tag barcode sequences are present and are specific to individual aliquots.
Release of tagged cDNA from aliquot-specified labeled nuclei
Dividing combined nuclei into samples and lysing the nuclei in each sample to release tagged cDNA molecules and form a lysate in each sample.
The claim coverage centers on nucleus-specific split-pool barcoding achieved via aliquot-specific RT primer barcode sequences associated with RT in fixed and permeabilized nuclei at below about 8°C, a shared 5′ overhang present at the cDNA 5′ end, followed by aliquot-specific nucleic acid tag barcode coupling defined by tag barcode sequences plus 3′ and/or 5′ hybridization sequences, and lysing to release tagged cDNA molecules into sample lysates.
Stated Advantages
Documented Applications
No documented applications found
Interested in licensing this patent?