Granted Patent B2

US 12,606,860 · App. 17/480,724 · Granted Apr 21, 2026

Inventors: Luigi Jhon Alvarado Martinez (Walnut Creek, CA); Eswar Prasad Ramachandran Iyer (Sunnyvale, CA); Jason Bell (Palo Alto, CA)

Assignee: 10X GENOMICS, INC.

C12Q1/6809C12Q1/6806C12Q1/6855C12Q1/6874

Abstract

Claims (33)

1 . A method for assaying ribonucleic acid (RNA) molecules, comprising:

(a) providing a cell or nucleus that comprises ribonucleic acid (RNA) molecules;

(b) depleting ribosomal RNA (rRNA) molecules from said RNA molecules in said cell or nucleus, to yield a plurality of remaining RNA molecules;

(c) fragmenting, in said cell or nucleus, said plurality of remaining RNA molecules to yield a plurality of RNA fragments;

(d) providing a plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a common barcode sequence; and

(e) using an RNA fragment of said plurality of RNA fragments and a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to generate a barcoded nucleic acid molecule.

2 . The method of claim 1 , wherein, in (e), said nucleic acid barcode molecule has, hybridized thereto, an RNA molecule, and wherein (e) further comprises performing a nucleic acid reaction using a thermostable group II intron reverse transcriptase (TGIRT).

3 . The method of claim 1 , wherein (e) further comprises: (i) conducting a nucleic acid reaction; and (ii) subsequent to said nucleic acid reaction, adding an adaptor molecule to said barcoded nucleic acid molecule.

4 . The method of claim 3 , wherein said adaptor molecule comprises a sequence comprising a 5′-App and 3′-blocker.

5 . The method of claim 3 , wherein said adding comprises ligating said adaptor molecule to said barcoded nucleic acid molecule using a 5′-App DNA/RNA ligase.

6 . The method of claim 3 , wherein said adaptor molecule comprises a splint oligonucleotide.

7 . The method of claim 6 , wherein said splint oligonucleotide comprises a random N-mer or a 3′ blocker.

8 . The method of claim 6 , wherein said adding comprises ligating said adaptor molecule to said barcoded nucleic acid molecule using a T4 DNA ligase.

9 . The method of claim 1 , wherein (e) further comprises conducting a nucleic acid reaction is performed using a T4KQ ligase.

10 . The method of claim 9 , further comprising, subsequent to said nucleic acid reaction, using said barcoded nucleic acid molecule or a derivative thereof in a reverse transcription reaction to generate an additional barcoded nucleic acid molecule comprising an adaptor sequence.

11 . The method of claim 10 , wherein said adaptor sequence comprises a template switching oligonucleotide.

12 . The method of claim 10 , wherein said adaptor sequence comprises a sequencing primer sequence, a reverse transcription primer sequence, and a uridine residue.

13 . The method of claim 12 , further comprising, subsequent to said reverse transcription reaction, subjecting said barcoded nucleic acid molecule to circularization, clean up, and linearization.

14 . The method of claim 13 , wherein said method comprises performing said circularization using a T4 DNA ligase, a splint oligonucleotide, or a single-stranded DNA ligase.

15 . The method of claim 14 , wherein said method comprises performing said circularization using said single-stranded DNA ligase, wherein said single-stranded DNA ligase is Circligase.

16 . The method of claim 12 , further comprising, prior to said reverse transcription, subjecting said barcoded nucleic acid molecule to (i) phosphorylation using a first enzyme, and (ii) ligation of a complement of said sequencing primer sequence using a second enzyme.

17 . The method of claim 16 , wherein said first enzyme is a T4 polynucleotide kinase and said second enzyme is a T4 RNA ligase.

18 . The method of claim 1 , wherein said cell or nucleus is a fixed and permeabilized cell or nucleus.

19 . The method of claim 1 , wherein said fragmenting comprises a chemical fragmentation process or an enzymatic fragmentation process.

20 . The method of claim 19 , wherein said chemical fragmentation process comprises using divalent metal cations and heating or using a reactive oxygen species.

21 . The method of claim 19 , further comprising, subsequent to said fragmentation process, cleaving a 3′ phosphate group from said RNA fragment.

22 . The method of claim 19 , wherein said enzymatic fragmentation process comprises using RNase III.

23 . The method of claim 1 , wherein said plurality of nucleic acid barcode molecules comprises a capture sequence.

24 . The method of claim 1 , wherein said plurality of remaining RNA molecules comprises non-poly-adenylated RNA molecules, wherein said RNA fragment is a fragment of a non-poly-adenylated RNA molecule of said non-poly-adenylated RNA molecules.

25 . The method of claim 24 , wherein said non-poly-adenylated RNA molecules comprises miRNA, long non-coding RNA, short non-coding RNA, tRNA, RNA isoforms, snoRNA, small RNA, piRNA, or circular RNA.

26 . The method of claim 1 , wherein said plurality of RNA fragments further comprises a poly-adenylated RNA fragment.

27 . The method of claim 1 , further comprising performing (d) within a partition, wherein said partition is a droplet.

28 . The method of claim 1 , further comprising performing (d) within a partition, wherein said partition is a well.

Assignments (1)

Continuity (3)

References Cited (400)