Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

Lee, J. H., Daugharthy, E. R., Scheiman, J., Kalhor, R., Ferrante, T. C., Terry, R., Turczyk, B. M., Yang, J. L., Lee, H. S., Aach, J., Zhang, K., Church, G. M. (2015) Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc, 10 (3). pp. 442-58. ISSN 1750-2799

URL: http://www.ncbi.nlm.nih.gov/pubmed/25675209
DOI: 10.1038/nprot.2014.191

Abstract

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Item Type: Paper
Subjects: Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA expression
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene expression
CSHL Authors:
Communities: CSHL Cancer Center Program > Cancer Genetics
CSHL Cancer Center Shared Resources > Animal Services
CSHL labs > Lee lab
CSHL Cancer Center Shared Resources > Animal Tissue and Imaging Service
Depositing User: Matt Covey
Date Deposited: 14 Oct 2015 19:11
Last Modified: 29 Oct 2015 20:18
PMCID: PMC4327781
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31922

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