DOI: 10.6084/m9.figshare.25685961
title: Xenium Renal Cell Carcinoma

description: We generated Xenium data from four clear-cell renal cell carcinoma (ccRCC) patient samples each consisting of tumor and adjacent tissue. A customized gene panel was used consisting of 280 genes provided by a base panel (10X's initial breast cancer gene panel) and 100 genes selected to target kidney-specific genes, T-cell subsets, and genes that were previously observed to show significant variation in expression in previously generated single-cell RNA-Seq data.This dataset was used it part to demonstrate an improved cell segmentation method <b>Proseg</b>, in the paper <i>Cell Simulation as Cell Segmentation</i>.Further details:The RCC FFPE blocks were provided by Northwest Biotrust, under a NWBiospecimens protocol, Seattle. NW BioTrust, a core service for patient consenting, and NWBioSpecimen, a core service for procurement and annotation of research biospecimens. The analysis was performed according to the IRB file/approval number NHS #6007-1061. No age or gender information is available. Informed written consent was obtained from each subject or each subject’s guardian.<br>Xenium allows customization of gene panels, which we took advantage of for this project. We selected 100 genes to add on to a standard breast cancer base panel of 280 genes. Fourteen of these were manually selected from prior interest and the remaining 86 were selected using an existing in an unsupervised manner using a prior scRNA-Seq dataset. Briefly, multinomial logistic regression was fit to cell type labels in this data and genes were prioritized by their absolute regression coefficient, as an approximation of how useful the gene is in distinguishing known cell types. The 100 add on genes were finalized in collaboration with 10X, removing some with very high expression risking optical crowding.We followed the Demonstrated Protocol Xenium in Situ Tissue Preparation Guide (CG000578 Rev A) to place samples on Xenium slides (10.45mm x 22.45mm). Deparaffinization and decrosslinking steps followed protocol CG000580 (Rev A). The Xenium In Situ Gene Expression User Guide (GC000582 Rev A) was used for the remaining workflow. The human breast base panel (280 genes) and a 100 gene custom add-on panel (See supplementary) were combined at the probe hybridization step. The slides were loaded in the Xenium Analyzer as directed by CG000584 (Rev A). Imaging was conducted in cycles on the instrument, capturing data across multiple Z-planes and fluorescence channels to build a spatial map of transcripts within selected scanned regions. For two samples we selected five regions within each tissue representing tumor and adjacent based on histology. We scanned the entire tissue as a single region for the second pair samples. The onboard analysis pipeline included image processing, nucleus and cell segmentation, RCA product image registration, transcript decoding, quality scoring, and deduplication. Quality scores were estimated using controls for calibration, ensuring the accuracy of the final output data. After decoding, duplicate transcripts were reconciled, and a cell-feature matrix was generated, allowing for further analysis and integration with existing datasets.