PMID- 25675209 OWN - NLM STAT- MEDLINE VI - 10 IP - 3 TI - Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. PG - 442-58 LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. PL - England TA - Nat Protoc JT - Nature protocols JID - 101284307 IS - 1750-2799 (Electronic) LID - 10.1038/nprot.2014.191 [doi] FAU - Lee, Je Hyuk AU - Lee JH AUID- ORCID: 0000000169477446 AD - Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. FAU - Daugharthy, Evan R AU - Daugharthy ER AD - 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. FAU - Scheiman, Jonathan AU - Scheiman J AD - 1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. FAU - Kalhor, Reza AU - Kalhor R AUID- ORCID: 0000000255587545 AD - Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. FAU - Ferrante, Thomas C AU - Ferrante TC AD - Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. FAU - Terry, Richard AU - Terry R AD - Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. FAU - Turczyk, Brian M AU - Turczyk BM AD - Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. FAU - Yang, Joyce L AU - Yang JL AD - Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. FAU - Lee, Ho Suk AU - Lee HS AD - Department of Electrical and Computer Engineering, University of California San Diego, California, USA. FAU - Aach, John AU - Aach J AD - Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. FAU - Zhang, Kun AU - Zhang K AUID- ORCID: 0000000275965224 AD - Department of Bioengineering, University of California San Diego, La Jolla, California, USA. FAU - Church, George M AU - Church GM AD - 1] Wyss Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. IS - 1750-2799 (Linking) RN - 0 (DNA, Complementary) RN - 0 (RNA, Messenger) SB - IM MH - DNA, Complementary/genetics MH - Fluorescence MH - Gene Expression Profiling/*methods MH - Microscopy, Confocal/methods MH - RNA, Messenger/*genetics MH - Sequence Analysis, RNA/*methods PMC - PMC4327781 DCOM- 20151019 LR - 20181113 DP - 2015 Mar DEP - 20150212 AB - 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.