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Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Accepted version
Peer-reviewed

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Authors

Herzog, Veronika A 
Neumann, Tobias 
Gapp, Katharina 
Zuber, Johannes 

Abstract

Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA. Total RNA isolated from a tissue of interest is then sequenced with SLAMseq, which introduces thymine to cytosine (T>C) conversions at the sites of the incorporated 4-thiouracil. The resulting sequencing reads are then mapped with the T>C-aware alignment software, SLAM-DUNK, which allows mapping of reads containing T>C mismatches. The number of T>C conversions per transcript is further analyzed to identify which transcripts are synthesized in the UPRT-expressing cells. Thus, our method, SLAM-ITseq (SLAMseq in tissue), enables cell-specific transcriptomics without laborious FACS-based cell sorting or biochemical isolation of the labeled transcripts used in TU tagging. In the murine tissues we assessed previously, this method identified ~5,000 genes that are expressed in a cell type of interest from the total RNA pool from the tissue. Any laboratory with access to a high-throughput sequencer and high-power computing can adapt this protocol with ease, and the entire pipeline can be completed in <5 d.

Description

Keywords

Animals, Female, Gene Expression Profiling, High-Throughput Nucleotide Sequencing, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Organ Specificity, Pentosyltransferases, Sequence Analysis, RNA, Thiouracil, Transcriptome

Journal Title

Nature Protocols

Conference Name

Journal ISSN

1754-2189
1750-2799

Volume Title

Publisher

Nature Publishing Group

Rights

All rights reserved
Sponsorship
Wellcome Trust (092096/Z/10/Z)
Wellcome Trust (104640/Z/14/Z)
Cancer Research UK (18583)
Cancer Research Uk (None)
Wellcome Trust (203144/Z/16/Z)
This work was supported by grants from Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) to E.A.M.; and a grant from the European Research Council (ERC-StG-338252 miRLIFE) to S.L.A. The IMP is generously supported by Boehringer Ingelheim. W.M. was supported by the Nakajima Foundation and St John’s College Benefactors’ Scholarship. K.G. was supported by a Swiss National Foundation postdoc mobility fellowship.