Single cell analyses

Patent metadata

Field	Value
Publication number	US-11767557-B2
Application number	US-201816213551-A
Country	US
Kind code	B2
Filing date	Dec 7, 2018
Priority date	Dec 7, 2017
Publication date	Sep 26, 2023
Grant date	Sep 26, 2023

Abstract

Modifications to both hardware and enzymatic reactions used in single cell analyses such as but not limited to Seq-well that enable significant increases in the yield of transcripts per cell, portability and ease of use, increased scalability of the assay, and linkage of transcript information to other measurements made in the picowell arrays are disclosed.

First claim

The invention claimed is:

1. A method for producing a library of nucleic acids containing universal primer sites on the 5′ and 3′ end from input nucleic acids comprising:
(a) providing a picowell array comprising a plurality of picowells,
(b) contacting input nucleic acids with a pool of capture oligonucleotides in a picowell of the plurality of picowells, each capture oligonucleotide in the picowell containing a 5′ universal primer site and a 3′ target binding site complementary to a nucleotide sequence in an input nucleic acid, wherein the input nucleic acids comprise RNA, and wherein the 3′ target binding site contained in the capture oligonucleotide comprises a poly(dT) sequence,
(c) adding a DNA polymerase and thereby extending the capture oligonucleotides hybridized to the input nucleic acids, to form first strand cDNA nucleic acids each comprising the 5′ universal primer site and a sequence that is complementary to one of the input nucleic acids,
(d) contacting the first strand cDNA nucleic acids with a pool of second strand priming oligonucleotides, each consisting of a 5′ universal primer site and a 3′ target binding site complementary to a nucleotide sequence in the first strand cDNA nucleic acid, wherein each 3′ target binding site of the second strand priming oligonucleotides consists of a random sequence,
(e) adding a DNA polymerase and thereby extending the second strand priming oligonucleotides, to form second strand cDNA nucleic acids comprising 5′ and 3′ universal primer sites that flank nucleotide sequences present in the input nucleic acids, and
(f) amplifying the second strand cDNA nucleic acids comprising 5′ and 3′ universal primer sites formed in step (e).

2. The method of claim 1 , wherein each capture oligonucleotide comprises a barcode present between the 5′ universal primer site and the 3′ target binding site.

3. The method of claim 1 , wherein the capture oligonucleotides are attached to a surface of a bead.

4. The method of claim 1 , wherein the input nucleic acids are derived from a single cell.

5. The method of claim 1 , wherein a crowding reagent is added in step (b), (c), (d) and/or (e).

6. The method of claim 1 , wherein the random sequence in the 3′ target binding site of the second strand priming oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long.

7. The method of claim 1 , further comprising pooling the content of a plurality of picowells into a single reaction volume after (b).

8. The method of claim 7 , wherein (d) and (e) are performed in the single reaction volume.

9. The method of claim 7 , wherein (c) is performed in the single reaction volume.

10. The method of claim 1 , wherein the DNA polymerase added in (e) lacks both 5′-3′ and 3′-5′ exonuclease activity.

11. The method of claim 4 , wherein the method further comprises loading the single cell to the picowell array, and wherein each picowell of the plurality of picowells comprises a functionalized surface comprising one or more nucleic acid barcodes.

12. The method of claim 11 , wherein each nucleic acid barcode is unique relative to all other nucleic acid barcodes in the array or to a subset of other nucleic acid barcodes in the array.

13. The method of claim 11 , wherein the location of each nucleic acid barcode in the array is known.

14. The method of claim 3 , wherein each of the capture oligonucleotides in the picowell further comprises a bead barcode that is the same in each capture oligonucleotide of the picowell.

15. The method of claim 14 , wherein the bead barcode identically labels the input nucleic acids derived from the same single cell.

16. The method of claim 3 , further comprising separating the second strand cDNA nucleic acids by base-mediated DNA denaturation.

17. The method of claim 1 , wherein the amplifying comprises PCR amplification.

Assignees

• Massachusetts Inst Technology

Inventors

• Love J Christopher
• Gierahn Todd Michael
• Shalek Alexander K
• Wadsworth Marc Havens
• Hughes Travis K

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• B01L3/5085 Primary

  for multiple samples, e.g. microtitration plates · CPC title

• C12N15/1068

  Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title