Department of Genetics, University of Cambridge, Cambridge, UK.
Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.
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.
分析细胞类型特异性转录组对于理解多细胞生物组织和器官的生物学特性至关重要。在这个方案扩展中，我们结合了一种先前开发的细胞类型特异性代谢 RNA 标记方法（硫代尿嘧啶 (TU) 标记）和一种新的 RNA 测序 (RNA-seq) 方法 SLAMseq（用于代谢 RNA 测序的硫 (SH) 连接烷基化）的检测标记转录本的方案。通过向表达细胞类型特异性尿嘧啶磷酸核糖基转移酶 (UPRT) 的转基因小鼠注射尿嘧啶类似物 4-硫代尿嘧啶，该酶是将 4-硫代尿嘧啶掺入新合成 RNA 所必需的，只有表达 UPRT 的细胞合成含硫 RNA。然后，从感兴趣的组织中分离总 RNA，并使用 SLAMseq 进行测序，该方法会在掺入的 4-硫代尿嘧啶位点将胸腺嘧啶转化为胞嘧啶 (T>C)。然后，将所得测序读段用 T>C 感知比对软件 SLAM-DUNK 进行映射，该软件允许对包含 T>C 错配的读段进行映射。进一步分析每个转录本的 T>C 转换数，以鉴定在 UPRT 表达细胞中合成的转录本。因此，我们的方法 SLAM-ITseq（组织中的 SLAMseq）无需费力的基于 FAC 的细胞分选或 TU 标记中使用的标记转录本的生化分离，即可实现细胞特异性转录组学分析。在我们之前评估的小鼠组织中，该方法从组织的总 RNA 池中鉴定出约 5000 个在感兴趣的细胞类型中表达的基因。任何拥有高通量测序仪和高性能计算能力的实验室都可以轻松地采用这种方案，并且整个方案可以在 <5 天内完成。
