| Tóm tắt: | 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.
|
|---|