Department of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8607, Japan.
Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-0054, Japan.
STAR Protoc. 2022 Apr 22;3(2):101346. doi: 10.1016/j.xpro.2022.101346. eCollection 2022 Jun 17.
Photo-isolation chemistry (PIC) enables isolation of transcriptome information from locally defined areas by photo-irradiation. Here, we present an optimized PIC protocol for formalin-fixed frozen and paraffin mouse sections and fresh-frozen mouse sections. We describe tissue section preparation and permeabilization, followed by reverse transcription using photo-caged primers. We then detail immunostaining and UV-mediated uncaging to the target areas, followed by linear amplification of uncaged cDNAs, library preparation, and quantification. This protocol can be applied to various animal tissue types. For complete details on the use and execution of this protocol, please refer to Honda et al. (2021).
光隔离化学（PIC）可通过光照射从局部定义的区域中分离转录组信息。在这里，我们提出了一个针对福尔马林固定的冷冻和石蜡小鼠切片以及新鲜冷冻的小鼠切片的优化 PIC 方案。我们描述了组织切片的制备和通透化，然后使用光笼引物进行逆转录。接下来，我们详细介绍了针对目标区域的免疫染色和 UV 介导的去笼，然后对去笼的 cDNA 进行线性扩增、文库制备和定量。该方案可应用于各种动物组织类型。如需详细了解该方案的使用和执行情况，请参阅 Honda 等人（2021 年）。
