SINGLE CELL ANALYSES

• US 20190218607A1
• Filed: 12/07/2018
• Published: 07/18/2019
• Est. Priority Date: 12/07/2017
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Love, J. Christopher, Gierahn, Todd Michael, Shalek, Alexander K., Wadsworth, Marc Havens, Hughes, Travis K.
• Granted Patent: US 11,767,557 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12N 15/1068 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 Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: G03F 7/2014 Contact or film exposure of light sensitive plates such as lithographic plates or circuit boards, e.g. in a vacuum frame

Abstract

This disclosure describes improvements 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, improved portability and ease of use, increased scalability of the assay, and linkage of transcript information to other measurements made in the picowell arrays.

Claims

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) contacting input nucleic acids with a pool of capture oligonucleotides, each capture oligonucleotide containing a 5′

universal primer site and a 3′

target binding site complementary to a nucleotide sequence in an input nucleic acid,

(b) adding a DNA polymerase and thereby extending the capture oligonucleotides hybridized to the input nucleic acids, to form first strand nucleic acids each comprising the 5′

universal primer site and a sequence that is complementary to one of the input nucleic acids,

(c) contacting the first strand nucleic acids with a pool of second strand priming oligonucleotides, each comprising a 5′

universal primer site and a 3′

target binding site complementary to a nucleotide sequence in the first strand nucleic acid, wherein each 3′

target binding site of the second strand priming oligonucleotides comprises a random sequence, and

(d) adding a DNA polymerase and thereby extending the second strand priming oligonucleotides, to form second strand nucleic acids comprising 5′ and

3′

universal primer sites that flank nucleotide sequences present in the input nucleic acids.

3. 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.

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

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

15. The method of claim 1, wherein the target binding sites in the pool of the second strand priming oligonucleotides further comprise a non-random sequence, wherein the non-random sequence is one of:

(a) a sequence that is complementary to specific sequences in the first strand nucleic acids, and (b) a sequence that prevents self-hybridization.

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

135. The method of claim 1, wherein the target binding sites in the pool of the second strand priming oligonucleotides comprise a random target binding sequence that is 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long.

2. (canceled)

4. (canceled)

6-10. -10. (canceled)

12-14. -14. (canceled)

16-18. -18. (canceled)

20-119. -119. (canceled)

120. A microwell device comprising:

(a) a photoresist film comprising a top surface, a bottom surface, and a plurality of through-holes from the top to the bottom surface, wherein each hole has a top opening on the top surface and a bottom opening on the bottom surface, and

(b) a porous bottom membrane in contact with the bottom surface of the photoresist film, wherein the bottom membrane has a flux rate of at least 0.1 mL/min/cm² as measured by the initial flux rate of water at 10 pounds per square inch (psi).

121. The device of claim 120, wherein the porous bottom membrane is a dry film membrane.

136. The device of claim 120, wherein the porous bottom membrane has a flux rate of 0.1-100 mL/min/cm².

137. The device of claim 120, wherein the porous bottom membrane has an average pore size of at least 50 nm.

138. The device of claim 120, wherein at least a portion of the plurality of the through-holes comprise a functional surface.

139. The device of claim 138, wherein the functional surface comprises a spatial barcode that is unique to the through-hole that comprises the functional surface.

140. The device of claim 120, wherein the top openings of the photoresist film have an average diameter of at least 15 microns and at most 100 microns.

122-126. -126. (canceled)

127. A method of cellular loading comprising,

(a) flowing a fluid sample comprising a plurality of cells through a microarray device, said device comprising, (i) a bottomless microwell array comprising a top surface, a bottom surface, and a plurality of through-holes from the top to the bottom surface, wherein each hole has a top opening on the top surface and a bottom opening on the bottom surface, and (ii) a porous bottom membrane in contact with the bottom surface of the microwell array, wherein the bottom membrane has a flux rate of at least 0.1 mL/min/cm² as measured by the initial flux rate of water at 10 pounds per square inch (psi),

(b) applying a pressure gradient from the top opening to the bottom opening of at least one of the plurality of through-holes, thereby loading a cell into said at least one through-hole, and

(c) retaining the cell at the bottom of the at least one through-hole by applying the pressure gradient, thereby loading cells in said through-holes in the microarray.

128. The method of claim 127, wherein a pressure gradient within the at least one through hole is reduced by the retained cell to a level insufficient to induce a loading of a second cell.

129. The method of claim 127, further comprising washing the cell or the microwell array when the microwell array is inverted.

131. The method of claim 127, wherein a plurality of beads are pre-loaded in the microwell array.

132. The method of claim 127, further comprising flowing a fluid sample comprising a plurality of beads.

141. The method of claim 127, further comprising inverting the microwell array while the cell is retained and reversing the inverted microwell array.

130. (canceled)

133-134. -134. (canceled)