Multiple Feature Integration With Next-Generation Three-Dimensional in Situ Sequencing
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsKarl A. Deisseroth, Ethan B. Richman
Life of this patent
Estimating the term…
Abstract
Provided herein are devices, methods, and systems for multiple feature integration with next-generation three-dimensional in situ sequencing of nucleic acids in cells in intact tissue. Biological samples contain many distinct types of molecular, cellular, anatomical, and experimental features. The disclosed methods allow simultaneous interrogation of multiple distinct features of a biological sample, including RNA features, anatomical features, exogenous barcodes, or other arbitrary experimental features such as in vivo measurements, which can be combined into single experimental readouts with next-generation in situ sequencing.
Claims95
A method of in situ sequencing of a target nucleic acid in a cell in an intact tissue in combination with cell barcoding, the method comprising:
- introducing into the cell in the intact tissue a nucleic acid comprising a sequence encoding a messenger RNA (mRNA) transcript comprising a 3′-untranslated region (3′-UTR) comprising a cell barcode and a poly-adenylation site, wherein the cell barcode is adjacent to the poly-adenylation site, wherein the cell expresses the mRNA transcript;
- measuring morphological or functional characteristics of the cell in the intact tissue;
- sequencing the barcode of the mRNA transcript;
- and
- performing in situ gene sequencing of the target nucleic acid in the cell in the intact tissue, wherein the cell barcode is used for assignment of in situ sequencing data to the measured morphological or functional characteristics of the cell.
Dependencies are inferred from the claim text.