Sequencing cell-type-specific transcriptomes with SLAM-ITseq

Nat Protoc. 2019 Aug;14(8):2261-2278. doi: 10.1038/s41596-019-0179-x. Epub 2019 Jun 26.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Female
  • Gene Expression Profiling / methods*
  • High-Throughput Nucleotide Sequencing
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Organ Specificity / genetics*
  • Pentosyltransferases / genetics
  • Pentosyltransferases / metabolism
  • Sequence Analysis, RNA / methods*
  • Thiouracil / analogs & derivatives
  • Thiouracil / chemistry
  • Thiouracil / metabolism
  • Transcriptome / genetics*


  • 4-thiouracil
  • Thiouracil
  • Pentosyltransferases
  • uracil phosphoribosyltransferase