title: Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.
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.
authors: Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors: Je Hyuk
authors: 0000000169477446
authors: JH
authors: Lee
authors: 1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors: Evan R
authors: ER
authors: Daugharthy
authors: 1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors: Jonathan
authors: J
authors: Scheiman
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors: Reza
authors: 0000000255587545
authors: R
authors: Kalhor
authors: Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors: Thomas C
authors: TC
authors: Ferrante
authors: Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors: Richard
authors: R
authors: Terry
authors: Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors: Brian M
authors: BM
authors: Turczyk
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors: Joyce L
authors: JL
authors: Yang
authors: Department of Electrical and Computer Engineering, University of California San Diego, California, USA.
authors: Ho Suk
authors: HS
authors: Lee
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors: John
authors: J
authors: Aach
authors: Department of Bioengineering, University of California San Diego, La Jolla, California, USA.
authors: Kun
authors: 0000000275965224
authors: K
authors: Zhang
authors: 1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors: George M
authors: GM
authors: Church
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger