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Interrogation of gene regulatory circuits in complex organisms requires precise and robust methods to label cell-types for profiling of target proteins in a tissue-specific fashion as well as data analysis to understand interconnections within the circuits. There are several strategies for obtaining cell-type and subcellular specific genome-wide data. We have developed a methodology, termed "biotagging" that uses tissue-specific, genetically encoded components to biotinylate target proteins, enabling in depth genome-wide profiling in zebrafish. We have refined protocols to use the biotagging approach that led to enhanced isolation of coding and non-coding RNAs from ribosomes and nuclei of genetically defined cell-types. The ability to study both the actively translated and transcribed transcriptome in the same cell population, coupled to genomic accessibility assays has enabled the study of cell-type specific gene regulatory circuits in zebrafish due to the high signal-to-noise achieved via its stringent purification protocol. Here, we provide detailed methods to isolate, profile and analyze cell-type specific polyribosome and nuclear transcriptome in zebrafish.

Original publication

DOI

10.1016/j.ymeth.2018.07.011

Type

Journal article

Journal

Methods

Publication Date

01/11/2018

Volume

150

Pages

24 - 31

Keywords

Cell-type specific in vivo biotinylation, Enhancer RNA, Nuclear transcriptome, Animals, Biotinylation, Cell Fractionation, Gene Expression Profiling, Gene Regulatory Networks, Polyribosomes, RNA, Staining and Labeling, Transcriptome, Zebrafish