Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues
| Autor: | Thomas C. Ferrante, George M. Church, Jonathan Scheiman, Joyce L. Yang, Brian M. Turczyk, Reza Kalhor, Kun Zhang, Richard C. Terry, Evan R. Daugharthy, Hosuk Lee, John Aach, Je-Hyuk Lee |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
DNA
Complementary Microscopy Confocal RNA localization Sequence Analysis RNA Gene Expression Profiling Fluorescent in situ sequencing RNA RNA-Seq Computational biology RNA integrity number Biology Molecular biology General Biochemistry Genetics and Molecular Biology Article Fluorescence Gene expression profiling Chimeric RNA Gene expression RNA Messenger |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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