NanoString GeoMx Digital Spatial Profiler whole-transcriptome spatial RNA dataset of CD4⁺ and CD20⁺ regions in melanoma tumor sections

This repository contains NanoString GeoMx Digital Spatial Profiler (DSP) whole-transcriptome spatial RNA profiling data generated from formalin-fixed paraffin-embedded (FFPE) tumor sections of five melanoma patients included in the paired single-cell RNA-seq cohort. Spatial transcriptomics was performed to investigate the localization and transcriptional states of CD4⁺ T cell–enriched and CD20⁺ B cell–enriched regions of interest (ROIs), and to assess their spatial relationships inside and outside the tumor bed. CD4⁺ ROIs were further classified as proximal or distal relative to CD20⁺ B cell regions to evaluate potential T–B cell interactions. Spatial profiling was performed using the NanoString GeoMx DSP Whole Transcriptome Atlas (WTA). FFPE tissue sections (5 µm) were processed according to manufacturer protocols, including antigen retrieval, proteinase K digestion, hybridization with WTA probes, and immunofluorescence staining for CD4 and CD20 to guide ROI selection. Regions of interest were defined using morphological markers and UV-photocleavage was used to release indexed oligonucleotide tags from selected areas. Libraries were generated by PCR amplification of released indexing oligos and sequenced on an Illumina NovaSeq platform. Digital count conversion (DCC) files were generated using the NanoString GeoMx DnD pipeline. Quality control and preprocessing were performed using GeoMx DSP Analysis Suite and the R package GeoMxTools. Probe-level filtering, segment filtering, and gene filtering were applied according to manufacturer-recommended criteria. Gene counts were normalized using upper quartile (Q3) normalization. Repository contents data_CD4CD20_segments.zip Raw.xlsx: raw digital counts per ROI Normalize data.xlsx: Q3-normalized gene expression matrix Annotation.xlsx: ROI-level annotations (patient ID, tissue location, CD4/CD20 enrichment, spatial classification, etc.) Annotation_Probe.xlsx: probe-level annotation and QC information images.zip Immunofluorescence-stained slide images used for ROI selection Visual indication of analyzed ROIs (CD4⁺ and CD20⁺ segments)