Multimodal readouts for quantifying and sequencing nucleic acids in single cells

US11414701B2 · kind B2 · utility

Assignees

• The Broad Institute, Inc. · US
• President and Fellows of Harvard College · US
• Massachusetts Institute of Technology · US

Inventors

• Fei Chen · Thạnh Trung, VN
• Jesse ENGREITZ · Cambridge, US
• Jamie Clive Marshall · Cambridge, US
• Vidya Subramanian · Somerville, US
• Sam Rodriques · Cambridge, US

Key dates

• Filing date: May 24, 2019
• Grant date: Aug 16, 2022
• Expiry date: Oct 30, 2039

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2600/16
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Provided herein are methods for generating single-cell molecular analysis comprising a) delivering one or more proximity dependent probes to a cell population, wherein each proximity dependent probe comprises a target binding region configured to bind a target RNA and a primer binding site region; b) linking bound proximity dependent probes; c) isolating single cells from the cell population in separate individual discrete volumes, the individual discrete volumes further comprising a primer pair and amplification reagents, wherein the primer pair binds to the primer binding sites of the ligation dependent probes, and wherein at least one primer comprises a barcode sequence that uniquely identifies the individual discrete volume; d) amplifying the ligated probes using the primer pair, wherein the barcode is incorporated into each resulting amplicon; and e) quantifying target RNAs in each individual cell based at least in part on sequencing the resulting amplicons.