Rna probing and amplification methods for single cell analysis on fixed cells

A method for determining the copy number of a nucleic acid target in a sample, comprising:

• contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target,
• extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target;
• barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target;
• obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target;
• and
• determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

A method for determining the spatial location and copy number of a nucleic acid target in a sample, comprising:

• contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label,
• wherein probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences;
• extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target;
• barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target;
• obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target;
• and
• for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.

A kit comprising:

• a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize a nucleic acid target, optionally the probing oligonucleotides comprise a predetermined spatial label;
• a coupling oligonucleotide comprising a 5′ complement of the coupling sequence and a 3′ complement of a capture sequence;
• a plurality of oligonucleotide barcodes, wherein the 3′ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, wherein the 5′ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence;
• a first primer capable of hybridizing to the first universal sequence, optionally further comprising a third universal sequence;
• amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, optionally further comprising a second universal sequence;
• a DNA ligase;
• extension reagents, optionally reverse transcription reagents, further optionally a reverse transcriptase and dNTPs;
• one or more fixing agents;
• one or more permeabilizing agents;
• a cross-linking agent;
• unfixing agent;
• lysis buffer;
• a plurality of cellular component-binding reagents, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets;
• a blocking reagent;
• one or more decoy oligonucleotides;
• and/or
• one or more blocking oligonucleotides.