GSM9514749: Bru.LC.3_33.1; Mus musculus; OTHER
Tissue Collection and Embedding Tissue specimens harvested from the experimental mouse models including B16-OVA subcutaneous tumors, AAV-induced GEMM liver tumors, HT29/E0771 lung metastases, and SMARTA splenic tissues were processed as either fresh frozen (FF) or formalin-fixed paraffin-embedded (FFPE) blocks. Fresh tissues were embedded in OCT compound immediately upon harvest, while fixed samples were preserved in PFA prior to paraffin embedding. To maintain RNA, DNA, and protein integrity for downstream spatial transcriptomics, all procedures from collection to embedding were conducted in a strict contamination-free environment. Suitability for analysis was confirmed by histological examination of adjacent sections. Sectioning and Slide Preparation For Perturb-DBiT profiling, both paraffin and OCT blocks were sliced into 7–10 µm thick sections. Sections were carefully mounted in the center of poly-L-lysine-coated glass slides (63478-AS, Electron Microscopy Sciences) to ensure proper alignment with the microfluidic device channels. FFPE Samples: Sectioning of archived FFPE liver and lung tissue blocks was performed by Yale Pathology Tissue Services (YPTS). Fresh Frozen Samples: Sectioning of frozen blocks was carried out in-house. Following preparation, all slides were stored at -80°C until the time of the experiment to prevent transcript degradation. Regulatory Compliance Tissue retrieval and distribution were conducted in accordance with approved protocols (Yale University Institutional Review Board IRB #1401013259) and under the oversight of the Tissue Resource Oversight Committee. All samples were handled in strict compliance with relevant institutional research policies and diagnostic requirements. The SEQuoia RiboDepletion Kit was used to remove rRNA and mitochondrial rRNA from the amplified cDNA, following the manufacturer's instructions. 20 ng of cDNA was used as the input amount, and three rounds of depletion were performed. 11 PCR cycles was sufficient to directly ligate sequencing primers, using a 100 μl system consisting of 50 μl 2X KAPA HiFi HotStart ReadyMix, ~42 μl solution from the rRNA removal step, 4 μl 10 μM Modified N501 Primer, and 4 μl 10 μM P7 Primer. The library product was purified using SPRIselect beads at a 0.8X ratio, according to the manufacturer's standard protocol.