SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting.

Wayo Matsushima,Katharina Gapp,Stefan L Ameres,Eric A Miska,Johannes Zuber,Veronika A Herzog,Tobias Neumann

doi:10.1242/dev.164640

Abstract

ABSTRACTCell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine and adipocytes in white adipose tissue. As this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.

Highlights

Animals consist of various organs, which are further composed of heterogeneous populations of highly specialised cells
During the reverse transcription step of RNA sequencing (RNA-seq) library preparation, a guanine (G), instead of an adenine (A), is basepaired to an alkylated 4-thiouracil leading to the thymine to cytosine base conversion (T>C) at the corresponding T position in the reads generated from the thio-RNA
One potential problem with the use of 4-thiouracil for tagging is that it could potentially be incorporated to RNA independent of external uracil phosphoribosyltransferase (UPRT) expression either by negligible endogenous Uprt activity or by an unknown alternative salvage pathway

Summary

Introduction

Animals consist of various organs, which are further composed of heterogeneous populations of highly specialised cells. To capture the transcriptome of a specific cell type, mechanical cell isolation methods such as fluorescence-activated cell sorting (FACS) or laser-capture microdissection (LCM) prior to RNA quantification have widely been used so far Combined with such cell-isolation methods, the recent advance in high-throughput RNA sequencing (RNA-seq) methods enables us to quantitate transcripts at single-cell resolution (Tang et al, 2010). Received 14 March 2018; Accepted 1 June 2018 closely transcriptomic data obtained from sorted cells reflect the state prior to cell sorting (Richardson et al, 2015) These cell-isolation methods are often time-intensive, involve laborious steps and lead to considerable cell death after isolation, which limits their applications to robust cells only

Methods

Results

Conclusion