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. (March 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
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: | March 2015 |
| Date Deposited: | 14 Oct 2015 19:11 |
| Last Modified: | 29 Oct 2015 20:18 |
| PMCID: | PMC4327781 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/31922 |
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