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Mixed-species RNA-seq for elucidating non-cell-autonomous control of gene transcription

Research output: Contribution to journalArticle

Original languageEnglish
Number of pages26
JournalNature Protocols
DOIs
Publication statusPublished - 24 Sep 2018

Abstract

Transcriptomic changes induced in one cell type by another mediate many biological processes in the brain and elsewhere; however, achieving artefact-free physical separation of cell types to study them is challenging and generally only allows for analysis of a single cell type. We describe an approach employing co-culture of distinct cell-types from different species, which enables physical cell sorting to be replaced by in silico RNA sequencing (RNA-seq) read sorting due to evolutionary divergence of mRNA sequence. As an exemplary experiment, we describe the co-culture of purified neurons, astrocytes, and microglia from different species (12–14 days). Following conventional RNA-seq, we then describe how to use our Python tool Sargasso (http://statbio.github.io/Sargasso/) to separate reads according to species and how to eliminate any artefacts borne out of imperfect genome annotation (10 hours). We show how this procedure, which requires no special skills beyond those that might normally be expected of wet-lab and bioinformatics researchers, enables the simultaneous transcriptomic profiling of different cell types, revealing the distinct influence of microglia on astrocytic and neuronal transcriptomes under inflammatory
conditions.

    Research areas

  • transcription, RNA-seq, mixed-species, tissue culture, neurons, astrocytes, microglia, bioinformatics, differential gene expressoin

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