Systems and methods for multiplexed measurements in single and ensemble cells

Patent metadata

Field	Value
Publication number	US-12377396-B2
Application number	US-201816967402-A
Country	US
Kind code	B2
Filing date	Dec 19, 2018
Priority date	Feb 5, 2018
Publication date	Aug 5, 2025
Grant date	Aug 5, 2025

Abstract

Provided are systems and methods that can combine T cell receptor sequencing (TCRseq) and Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq), and/or respective aspects thereof. Further provided are systems and methods that can combine ATAC-seq and perturbation sequencing (Perturb-seq), and/or respective aspects thereof.

First claim

What is claimed is:

1. A method of processing immune cells, comprising:
(a) isolating a single immune cell comprising genomic deoxyribonucleic acid (gDNA) and a plurality of messenger ribonucleic acid (mRNA) molecules;
(b) tagmenting one or more regions of accessible chromatin in the gDNA of the single immune cell to produce a plurality of tagged gDNA fragments in a tagmentation reaction, wherein the tagmentation reaction comprises cleaving and tagging the gDNA in the same reaction;
(c) adding a primer, reverse transcriptase, and dNTPs to the single immune cell to reverse transcribe the plurality of mRNA molecules of the single immune cell to produce a plurality of complementary DNA (cDNA) molecules, wherein said cDNA molecules comprise sequences that correspond to a V(D)J region of a genome of the single immune cell;
(d) sequencing the plurality of cDNA molecules and the plurality of tagged gDNA fragments that were both produced from the single immune cell to generate a plurality of sequencing reads; and
(e) generating an epigenetic map of the one or more regions of accessible chromatin of the gDNA and the sequences that correspond to the V(D)J region of the single immune cell by correlating:
one or more sequencing reads of the plurality of tagged gDNA fragments or amplicons thereof; and
one or more sequencing reads of the plurality of cDNA molecules or amplicons thereof.

2. The method of claim 1 , wherein said plurality of immune cells comprises T cells or B cells.

3. The method of claim 1 , wherein (a) isolating the single immune cell comprises capturing the single immune cell in a partition.

4. The method of claim 3 , wherein said partition comprises a chamber, a well, or a droplet.

5. The method of claim 1 , wherein (a) isolating the single cell comprises flow cytometry sorting or magnetic cell sorting.

6. The method of claim 1 , further comprising lysing said single immune cell.

7. The method of claim 1 , wherein each of said tagged gDNA fragments comprises one or more adapters.

8. The method of claim 7 , wherein said one or more adapters comprise an adapter sequence.

9. The method of claim 1 , wherein said primer comprises a sequence specific to or targeted for a sequence of said plurality of mRNA molecules.

10. The method of claim 1 , wherein said plurality of mRNA molecules comprise T cell receptor alpha (TRA) or T cell receptor beta (TRB) mRNA molecules.

11. The method of claim 10 , wherein (c) comprises contacting the plurality of mRNA molecules with a plurality of primers, wherein said plurality of primers comprises a sequence specific to or targeted for a sequence encoding a constant region of said TRA or TRB mRNA molecules.

12. The method of claim 1 , wherein said plurality of mRNA molecules comprise T cell receptor alpha (TRA) and T cell receptor beta (TRB) mRNA molecules.

13. The method of claim 1 , further comprising contacting said plurality of tagged gDNA fragments and said plurality of cDNA molecules with a plurality of primers and a polymerase to generate tagged gDNA fragment amplicons or cDNA molecule amplicons.

14. The method of claim 13 , wherein each of at least a subset of said plurality of primers comprises a sequence specific to or targeted for a sequence encoding a constant region or variable region of a T cell receptor (TCR).

15. The method of claim 13 , wherein each of at least a subset of said plurality of primers comprises a sequence specific to or targeted for a sequence encoding a constant region and variable region of a T cell receptor (TCR).

16. The method of claim 1 , further comprising contacting said plurality of tagged gDNA fragments and said plurality of cDNA molecules with a plurality of primers and a polymerase to generate tagged gDNA fragment amplicons and cDNA molecule amplicons.

17. The method of claim 1 , further comprising generating amplicons from said plurality of tagged gDNA fragments and said plurality of cDNA molecules, wherein said amplicons each comprise a barcode sequence that identifies said single immune cell.

18. The method of claim 17 , wherein said amplicons each comprising said barcode sequence that identifies said single immune cell are generated from other amplicons of said plurality of tagged gDNA fragments or said plurality of cDNA molecules.

19. The method of claim 17 , further comprising sequencing said amplicons.

20. The method of claim 1 , further comprising, subsequent to (b), terminating said tagmentation reaction.

21. The method of claim 20 , wherein said terminating comprises contacting said single immune cell with a chelating agent.

22. The method of claim 21 , wherein said chelating agent is selected from the group consisting of: ethylenediamine tetraacetatic acid (EDTA), nitriloacetic acid (NTA), and diethylenetriamine pentaacetic acid (DTPA).

23. The method of claim 1 , wherein said tagmentation reaction comprises using a detergent.

24. The method of claim 23 , wherein said detergent comprises a non-ionic surfactant.

25. The method of claim 24 , wherein said detergent is an ethoxylated nonylphenol.

26. The method of claim 1 , wherein the tagmentation reaction comprises cleaving and tagging the gDNA using an insertional enzyme complex comprising a transposase and one or more one or more adapters.

27. The method of claim 1 , wherein each mRNA molecule comprises an immune cell receptor comprising the V(D)J region.

28. The method of claim 27 , wherein the immune cell receptor is a TCR or a B cell Receptor (BCR).

Assignees

• Univ Leland Stanford Junior

Inventors

• Saligrama Naresha
• Satpathy Ansuman
• Chang Howard Y
• Davis Mark M

Classifications

• C12Q2537/165

  Mathematical modelling, e.g. logarithm, ratio · CPC title

• C12Q1/6874

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

• C12N15/1096 Primary

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

• B01J2219/00722

  Nucleotides · CPC title

• B01J2219/00596

  Solid-phase processes · CPC title