Orthotopic 4T1 tumors spatial transcriptomics

4T1 tumors, 4 weeks after implantation were excised and analyzed using the 10X Visium technology. FFPE tissue blocks were cut down to approximately 6.5 mm x 6.5 mm in size and sectioned onto the capture areas of the Visium slide according to 10X Genomics Tissue Preparation Guide CG000408. Slides were then deparaffinized and H&E-stained according to the 10X Genomics Demonstrated Protocol CG000409 and imaged at the Animal & Tissue Imaging Core Facility at CSHL. After imaging, the tissue was decrosslinked and probe hybridization with the Visium Mouse Transcriptome Probe Kit (PN-1000365, 10X Genomics) was performed overnight at 50°C. Probe ligation, probe release and extension, and library preparation were performed according to manufacturer’s instructions, and final libraries were sequenced on a NextSeq 500 to target 20,000 reads per spot. Sequencing data were processed with Space Ranger version 2.0.0 (10X Genomics).

将植入4周后的4T1肿瘤组织切除，采用10X Visium技术（10X Visium）开展分析。将福尔马林固定石蜡包埋（FFPE，Formalin-Fixed Paraffin-Embedded）组织块切割至约6.5 mm × 6.5 mm大小，依照10X Genomics组织制备指南CG000408，将其切片至Visium玻片的捕获区域。随后按照10X Genomics示范方案CG000409对玻片进行脱蜡与苏木精-伊红（H&E）染色，并在冷泉港实验室（CSHL，Cold Spring Harbor Laboratory）动物与组织成像核心设施完成成像。成像完成后，对组织进行解交联处理，并使用Visium小鼠转录组探针试剂盒（PN-1000365，10X Genomics）在50℃条件下孵育过夜以完成探针杂交。探针连接、探针释放与延伸以及文库制备均依照制造商的操作指南进行，最终构建的文库在NextSeq 500测序平台上进行测序，目标为每个捕获位点获取20000条读段。测序数据采用Space Ranger 2.0.0版本（10X Genomics）进行处理。