Sequencing cell-type-specific transcriptomes with SLAM-ITseq

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.

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Fig. 1: Flow chart of the SLAM-ITseq protocol.
Fig. 2: Representative spectrophotometry results of 4-thiouracil before and after IAA treatment.
Fig. 3: T>C fraction comparison between Cre+ and Cre brain (Tie2:Cre, endothelial-specific Cre line).

Data availability

For replication of the example results shown in the protocol, use our published dataset. FASTQ files from SLAM-ITseq and metadata related to these are available from the ArrayExpress database at EMBL-EBI (https://www.ebi.ac.uk/arrayexpress/) under accession no. E-MTAB-6353.

Change history

  • 11 July 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank K. Harnish for high-throughput sequencing support, B. Reichholf and P. Bhat for technical support, and Wellcome Sanger Institute Research Support Facility staff for mouse maintenance and experimental support. 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.

Author information

W.M. and E.A.M. conceived and designed the study; W.M. wrote the manuscript; K.G., T.N., S.L.A., and E.A.M. reviewed and edited the manuscript; W.M. and V.A.H. performed the experiments; T.N. developed the SLAM-DUNK software and advised on the analyses; W.M. analyzed the data; K.G. administered the animal experiments and advised on the study design; S.L.A., J.Z., and E.A.M. provided expertise and feedback.

Correspondence to Eric A. Miska.

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Competing interests

E.A.M. is the founder and director of STORM Therapeutics Ltd.

Additional information

Peer review information: Nature Protocols thanks David Barrass, Charles G. Danko and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Protocol to which this is an extension

Gay, L., Karfilis, K. V., Miller. M. R., Doe, C. Q. & Stankunas, K. Nat. Protoc. 9, 410–420 (2014): https://doi.org/10.1038/nprot.2014.023

Key references using this protocol

Matsushima, W. et al. Development 145, dev164640 (2018): https://doi.org/10.1242/dev.164640

Herzog, V. A. et al. Nat. Methods 14, 1198–1204 (2017): https://doi.org/10.1038/nmeth.4435

Ameres, S., Herzog, V. A. & Reichholf, B. Protocol Exchange (2017): https://doi.org/10.1038/protex.2017.105

Muhar, M. et al. Science 360, 800–805 (2018): https://doi.org/10.1126/science.aao2793

Neumann, T. et al. BMC Bioinformatics 20, 258 (2019): https://doi.org/10.1186/s12859-019-2849-7.

This protocol is an extension to: Nat. Protoc. 9, 410–420 (2014), doi:10.1038/nprot.2014.023

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