Xenium data for a comparison of spatial transcriptomics technologies for medulloblastoma cryosections

10X Genomics Xenium data from human Medulloblastoma samples. The data was acuired in the course of a study performing a comparison of four imaging-based ST methods – RNAscope HiPlex, Molecular Cartography, MERFISH/Merscope, and Xenium – together with sequencing-based ST (Visium). These technologies were used to study cryosections of medulloblastoma with extensive nodularity (MBEN), a tumor chosen for its distinct microanatomical features. Our analysis reveals that automated imaging-based ST methods are well suited to delineating the intricate MBEN microanatomy, capturing cell-type-specific transcriptome profiles. We devise approaches to compare the sensitivity and specificity of the different methods together with their unique attributes to guide method selection based on the research aim. Furthermore, we demonstrate how reimaging of slides after the ST analysis can markedly improve cell segmentation accuracy and integrate additional transcript and protein readouts to expand the analytical possibilities and depth of insights. This study highlights key distinctions between various ST technologies and provides a set of parameters for evaluating their performance. Our findings aid in the informed choice of ST methods and delineate approaches for enhancing the resolution and breadth of spatial transcriptomic analyses, thereby contributing to advancing ST applications in solid tumor research. Cryosections from brain tissue of human patients with medullonlastoma with extensive nodularity of 10 µm thickness were obtained using a Cryostar NX50 (Epredia) cryostat at a cutting temperature of -15 °C. Tissues were sectioned into 10 µm slices, and four samples were placed on a single Xenium slide. After sectioning, the slides were stored at -80°C for less than two weeks. On the day of the experiment, the tissue was fixed with PFA according to the manufacturer's protocol. Tissues were permeabilized with SDS, incubated in 70% ice-cold methanol, and washed with PBS. Hybridization of the human generic brain panel with 70 add-on genes (Supplementary Dataset 1) was performed at 50°C in a Bio-Rad C1000 touch cycler for 20 hours. The washing, ligation, and amplification steps were carried out according to the manufacturer’s instructions. ROIs were selected based on the tissue area, excluding non-tissue-covered tiles. Each transcript was imaged in a bright state five times across 60 cycle-channels (15 cycles x 4 channels). After the run on the Xenium analyzer, slides were removed, and buffer was exchanged with PBS-T for further storage at 4°C.