Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues v1

• DOI: 10.17504/protocols.io.mgsc3we
• Publication Date: 2018-03-23T18:41:41Z
• Authors: Je Hyuk Lee; Evan R. Daugharthy; Jonathan Scheiman; Reza Kalhor; Thomas C. Ferrante; Richard Terry; Brian M. Turczyk; Joyce L. Yang; Ho Suk Lee; John Aach; Kun Zhang; and George M. Church
• Keywords: Amplicon; RNA-Seq

RNA sequencing measures the quantitative change in gene expression over whole transcriptome, but it lacks spatial context. On other hand, situ hybridization provides location of expression, only for a small number genes. Here we detail protocol genome-wide profiling fixed cells and tissues, which is converted into cross-linked cDNA amplicons sequenced manually on confocal microscope. Unlike traditional RNA-seq our method enriches context-specific transcripts house-keeping and/or structural RNA, preserves tissue architecture localization studies. Our written researchers experienced cell microscopy with minimal computing skills. Library construction can be completed within 14 d, image analysis requiring an additional 2 d.