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

被引：383

作者：

Lee, Je Hyuk ^{[1
]}

Daugharthy, Evan R. ^{[1
,2
,3
]}

Scheiman, Jonathan ^{[1
,2
]}

Kalhor, Reza ^{[2
]}

Ferrante, Thomas C. ^{[1
]}

Terry, Richard ^{[1
]}

Turczyk, Brian M. ^{[1
]}

Yang, Joyce L. ^{[2
]}

Lee, Ho Suk ^{[4
]}

Aach, John ^{[2
]}

Zhang, Kun ^{[5
]}

Church, George M. ^{[1
,2
]}

机构：

[1] Harvard Univ, Sch Med, Wyss Inst, Boston, MA 02115 USA

[2] Harvard Univ, Sch Med, Dept Genet, Boston, MA USA

[3] Harvard Univ, Sch Med, Dept Syst Biol, Boston, MA USA

[4] Univ Calif San Diego, Dept Elect & Comp Engn, San Diego, CA 92103 USA

[5] Univ Calif San Diego, Dept Bioengn, La Jolla, CA 92093 USA

来源：

NATURE PROTOCOLS | 2015年 / 10卷 / 03期

基金：

美国国家卫生研究院; 美国国家科学基金会;

关键词：

ESCHERICHIA-COLI; SINGLE CELLS; PRIMED RCA; AMPLIFICATION; MOLECULES; SEQ; TRANSCRIPTOMICS; REVEALS; PROTEIN; GENOME;

D O I：

10.1038/nprot.2014.191

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：442 / 458

页数：17

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