Xenium In Situ enables researchers to characterize RNAs in cells and tissues with subcellular resolution. Users can choose among several pre-designed panels, or design add-on custom or standalone custom panels for human and mouse. This Technical Note explains standalone custom panel design for species beyond those currently offered by 10x Genomics ("species standalone custom"), as well as advanced custom designs. Potential advanced custom applications include isoforms, gene fusions, viral or bacterial sequences, protein tags, fluorescent reporters, and transgenes. Types of customization possible, the basic structure of Xenium probes, and how users can select their own RNA-binding domain sequences are covered here. To submit an advanced custom design request, researchers must submit a CSV file, described below, to 10x Genomics. Before creating this file, users are advised to consider these guidelines for probe design. Although pre-designed panels have been extensively tested by 10x, species standalone custom and advanced customization are not fully supported — 10x Genomics will support adding these probes to the panel, but cannot guarantee that these probes will successfully detect their targets.