Highly multiplexed subcellular RNA sequencing in situ

Science. 2014 Mar 21;343(6177):1360-3. doi: 10.1126/science.1250212. Epub 2014 Feb 27.


Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Cell Line
  • Cells, Cultured
  • DNA, Complementary
  • Fluorescence
  • Gene Expression Profiling / methods*
  • Humans
  • Induced Pluripotent Stem Cells
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis
  • Transcription Initiation Site
  • Transcriptome*
  • Wound Healing


  • DNA, Complementary
  • RNA, Messenger

Associated data

  • GEO/GSE54733
  • GEO/GSM313643
  • GEO/GSM313646
  • GEO/GSM313657