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Sequencing Cell-Type-Specific Transcriptomes with SLAM-ITseq

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June 26, 2019

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

Nature Protocols 14, 2261–2278 (2019)

Sequencing cell-type-specific transcriptomes with SLAM-ITseqSLAM-ITseq design. Schematic of how SLAM-ITseq works. Cre is expressed in cells in which a cell type-specific promoter (Pcell) is activated and removes the floxed GFP-coding sequence followed by simian virus 40 polyadenylation sequence (SV40) between chicken β-actin promoter (CA) and UPRT-coding region, resulting in UPRT expression in the cells. When the mice are exposed to 4-thiouracil, only those UPRT-expressing cells (shown in yellow) can convert 4-thiouracil to 4-thio-UMP to synthesise thio-RNA. RNA is extracted from entire tissue without cell sorting, and the labelled RNA that is synthesised in the cells of interest is identified by finding T>C containing reads using SLAMseq.
[Figure/ Matsushima W , Herzog V A , Neumann T , et al. SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting[J]. Development, 2018, 145(13):dev.164640.]


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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