Deterministic barcoding for spatial omics sequencing

1. A method, comprising:

(a) delivering to a region of interest in a fixed section of a mammalian tissue mounted on a substrate a first set of barcoded polynucleotides that bind to nucleic acids of the fixed tissue section, wherein the first set of barcoded polynucleotides is delivered through a first microfluidic device clamped to the region of interest, wherein the first microfluidic device comprises variable width microchannels, each having (i) an inlet port and an outlet port, (ii) a width of 50-150 μm at the inlet port and at the outlet port, and (iii) a width of 10-50 μm at the region of interest;

(b) delivering to the region of interest reverse transcription reagents to produce cDNAs linked to barcoded polynucleotides of the first set;

(c) delivering to the region of interest a second set of barcoded polynucleotides, wherein the second set of barcoded polynucleotides is delivered through a second microfluidic device clamped to the region of interest, wherein the second microfluidic device comprises variable width microchannels, each having (i) an inlet port and an outlet port, (ii) a width of 50-150 μm at the inlet port and at the outlet port, and (iii) a width of 10-50 μm at the region of interest, wherein the microchannels of the second microfluidic device are perpendicular to the microchannels of the first microfluidic device;

(d) delivering to the region of interest ligation reagents to join barcoded polynucleotides of the first set to barcoded polynucleotides of the second set;

(e) imaging the region of interest to produce a sample image;

(f) delivering to the region of interest lysis buffer or denaturation reagents to produce a lysed or denatured tissue sample; and

(g) extracting cDNA from the lysed or denatured tissue sample.

2. The method of claim 1 further comprising sequencing the cDNA to produce cDNA reads.

3. The method of claim 1 , wherein the first and/or second microfluidic device is fabricated from polydimethylsiloxane (PDMS).

4. The method of claim 1 , wherein each microchannel of the first and second microfluidic device has a width of 10 μm and a height of 12-15 μm, a width of 25 μm and height of 17-22 μm, or a width of 50 μm and a height of 20-100 μm at the region of interest.

5. The method of claim 1 , wherein delivery of the first set of barcoded polynucleotides is delivered through the first microfluidic device using a negative pressure system and/or delivery of the second set of barcoded polynucleotides is delivered through the second microfluidic device using a negative pressure system.

6. The method of claim 1 , wherein the lysis buffer or denaturation reagents are delivered directly to the tissue section.

7. The method of claim 1 , wherein the barcoded polynucleotides of the first set comprise a ligation linker sequence, a spatial barcode sequence, and a polyT sequence.

8. The method of claim 7 , wherein the barcoded polynucleotides of the second set comprise a ligation linker sequence, a spatial barcode sequence, a unique molecular identifier (UMI) sequence, and a first PCR handle end sequence.

9. The method of claim 8 , wherein (i) the barcoded polynucleotides of the second set are bound to a universal ligation linker, or (ii) the method further comprises delivering to the fixed tissue section a universal ligation linker sequence, wherein the universal ligation linker comprises a sequence complementary to the ligation linker sequence of the barcoded polynucleotides of the first set and comprises a sequence complementary to the ligation linker sequence of the barcoded polynucleotides of the second set.

10. The method of claim 1 , wherein the first and/or second set of barcoded polynucleotides comprises at least 50 barcoded polynucleotides.

11. The method of claim 1 , wherein the nucleic acids of the fixed tissue section are selected from (i) ribonucleic acids (RNAs), and (ii) deoxyribonucleic acids (DNAs).

12. The method of claim 11 , wherein the nucleic acids of the fixed tissue section are messenger RNAs.

13. The method of claim 11 , wherein the nucleic acids of the fixed tissue section are genomic DNAs.

14. The method of claim 1 , wherein the imaging is with an optical or fluorescence microscope.

15. The method of claim 1 , wherein the first microfluidic device comprises at least 5 variable width microchannels and the second microfluidic device comprises at least 5 variable width microchannels.

16. The method of claim 15 , wherein the first microfluidic device comprises 5-100 variable width microchannels and the second microfluidic device comprises 5-100 variable width microchannels.

17. A method, comprising:

(a) delivering to a region of interest in a fixed section of a mammalian tissue mounted on a substrate binder-DNA tag conjugates that comprise (i) a binder molecule that specifically binds to a protein of interest and (ii) a DNA tag, wherein the DNA tag comprises a binder barcode and a polyA sequence;

(b) delivering to the region of interest a first set of barcoded polynucleotides that bind to nucleic acids of the fixed tissue section, wherein the first set of barcoded polynucleotides is delivered through a first microfluidic device clamped to the region of interest, wherein the first microfluidic device comprises variable width microchannels, each having (i) an inlet port and an outlet port, (ii) a width of 50-150 μm at the inlet port and at the outlet port, and (iii) a width of 10-50 μm at the region of interest;

(c) delivering to the region of interest reverse transcription reagents to produce cDNAs linked to barcoded polynucleotides of the first set;

(d) delivering to the region of interest a second set of barcoded polynucleotides, wherein the second set of barcoded polynucleotides is delivered through a second microfluidic device clamped to the region of interest, wherein the second microfluidic device comprises variable width microchannels, each having (i) an inlet port and an outlet port, (ii) a width of 50-150 μm at the inlet port and at the outlet port, and (iii) a width of 10-50 μm at the region of interest, wherein the microchannels of the second microfluidic device are perpendicular to the microchannels of the first microfluidic device;

(e) delivering to the region of interest ligation reagents to join barcoded polynucleotides of the first set to barcoded polynucleotides of the second set;

(f) imaging the region of interest to produce a sample image;

(g) delivering to the region of interest lysis buffer or denaturation reagents to produce a lysed or denatured tissue sample; and

(h) extracting cDNA from the lysed or denatured tissue sample.

18. The method of claim 17 further comprising sequencing the cDNA to produce cDNA reads, and constructing a spatial molecular expression map of the fixed tissue section by matching the binder-DNA tag conjugates to corresponding cDNA reads.

19. The method of claim 18 further comprising identifying the anatomical location of the nucleic acids by correlating the spatial molecular expression map to the sample image.

20. The method of claim 17 , wherein the binder molecule is an antibody.

21. The method of claim 20 , wherein the antibody is selected from the group consisting of: whole antibodies, Fab antibody fragments, F(ab′) 2 antibody fragments, monospecific Fab 2 fragments, bispecific Fab 2 fragments, trispecific Fab 3 fragments, single chain variable fragments (scFvs), bispecific diabodies, trispecific diabodies, scFv-Fc molecules, and minibodies.

22. The method of claim 17 , wherein the first microfluidic device comprises at least 5 variable width microchannels and the second microfluidic device comprises at least 5 variable width microchannels.

23. The method of claim 22 , wherein the first microfluidic device comprises 5-100 variable width microchannels and the second microfluidic device comprises 5-100 variable width microchannels.