Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues
Author
Lee, Je Hyuk
Daugharthy, Evan R
Terry, Richard
Turczyk, Brian M
Yang, Joyce L
Lee, Ho Suk
Zhang, Kun
Note: Order does not necessarily reflect citation order of authors.
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https://doi.org/10.1038/nprot.2014.191Metadata
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Lee, Je Hyuk, Evan R Daugharthy, Jonathan Scheiman, Reza Kalhor, Thomas C Ferrante, Richard Terry, Brian M Turczyk, et al. 2015. “Fluorescent in Situ Sequencing (FISSEQ) of RNA for Gene Expression Profiling in Intact Cells and Tissues.” Nature Protocols 10 (3) (February 12): 442–458. doi:10.1038/nprot.2014.191.Abstract
RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, 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 house-keeping 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.Other Sources
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327781/Terms of Use
This article is made available under the terms and conditions applicable to Open Access Policy Articles, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#OAPCitable link to this page
http://nrs.harvard.edu/urn-3:HUL.InstRepos:37165396
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