Single Cell Systems Biology Laboratory, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, 266100, China.
Department of Orthopedics, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China.
Commun Biol. 2024 Aug 20;7(1):1020. doi: 10.1038/s42003-024-06694-5.
Expression profiling in spatially defined regions is crucial for systematically understanding tissue complexity. Here, we report a method of photo-irradiation for in-situ barcoding hybridization and ligation sequencing, named PBHL-seq, which allows targeted expression profiling from the photo-irradiated region of interest in intact fresh frozen and formalin fixation and paraffin embedding (FFPE) tissue samples. PBHL-seq uses photo-caged oligodeoxynucleotides for in situ reverse transcription followed by spatially targeted barcoding of cDNAs to create spatially indexed transcriptomes of photo-illuminated regions. We recover thousands of differentially enriched transcripts from different regions by applying PBHL-seq to OCT-embedded tissue (E14.5 mouse embryo and mouse brain) and FFPE mouse embryo (E15.5). We also apply PBHL-seq to the subcellular microstructures (cytoplasm and nucleus, respectively) and detect thousands of differential expression genes. Thus, PBHL-seq provides an accessible workflow for expression profiles from the region of interest in frozen and FFPE tissue at subcellular resolution with areas expandable to centimeter scale, while preserving the sample intact for downstream analysis to promote the development of transcriptomics.
在空间上定义的区域进行表达谱分析对于系统地理解组织复杂性至关重要。在这里，我们报告了一种光照射原位条形码杂交和连接测序的方法，称为 PBHL-seq，它允许从完整的新鲜冷冻和福尔马林固定石蜡包埋（FFPE）组织样品中感兴趣的光照射区域进行靶向表达谱分析。PBHL-seq 使用光笼寡核苷酸进行原位反转录，然后对 cDNA 进行空间靶向条形码标记，从而创建光照射区域的空间索引转录组。我们通过将 PBHL-seq 应用于 OCT 包埋的组织（E14.5 小鼠胚胎和小鼠大脑）和 FFPE 小鼠胚胎（E15.5），从不同区域中恢复了数千个差异富集的转录本。我们还将 PBHL-seq 应用于亚细胞微结构（分别为细胞质和细胞核），并检测到数千个差异表达基因。因此，PBHL-seq 提供了一种可访问的工作流程，可用于从冷冻和 FFPE 组织中的感兴趣区域进行表达谱分析，具有亚细胞分辨率，并且可扩展到厘米级别的区域，同时保持样品完整，用于下游分析，以促进转录组学的发展。
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