# Protocol to evaluate mouse brain spatial cell type-resolved transcriptomic discoveries using 10× Visium spatial transcriptomics and FLEX scRNA-seq

## TL;DR

This paper provides a detailed protocol for using spatial transcriptomics and single-cell RNA sequencing to study gene expression in mouse brains at different ages.

## Contribution

The paper introduces a combined workflow using 10× Visium and FLEX scRNA-seq for spatially resolved transcriptomic analysis in mouse brain tissue.

## Key findings

- The protocol enables the collection and processing of mouse brain tissue for spatial and single-cell RNA sequencing.
- It includes steps for tissue staining, fixation, dissociation, and storage for downstream analysis.
- The method supports multi-age studies to investigate transcriptional changes in brain regions.

## Abstract

Understanding changes in gene expression and cell-cell signaling among spatial regions in diseased tissues adds critical biological information to understanding mechanisms. Here, we present a protocol to investigate molecular transcriptional drivers within intact murine tissue using 10× Genomics Visium spatial transcriptomics and 10× Genomics FLEX single-cell RNA sequencing (scRNA-seq) data. We describe steps for collecting mouse brain tissue from multiple ages, processing samples, and mounting the tissue. We then detail procedures for staining, FLEX tissue section collection, fixation, dissociation, and cell storage.

For complete details on the use and execution of this protocol, please refer to Burns et al.1

•Mouse brain collection at multiple time points for spatial transcriptomics and scRNA-seq•Steps for fresh frozen spatial tissue processing with Visium and FLEX scRNA-seq•Processing of postnatal day 0 mouse brain tissue for sequencing

Mouse brain collection at multiple time points for spatial transcriptomics and scRNA-seq

Steps for fresh frozen spatial tissue processing with Visium and FLEX scRNA-seq

Processing of postnatal day 0 mouse brain tissue for sequencing

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Understanding changes in gene expression and cell-cell signaling among spatial regions in diseased tissues adds critical biological information to understanding mechanisms. Here, we present a protocol to investigate molecular transcriptional drivers within intact murine tissue using 10× Genomics Visium spatial transcriptomics and 10× Genomics FLEX single-cell RNA sequencing (scRNA-seq) data. We describe steps for collecting mouse brain tissue from multiple ages, processing samples, and mounting the tissue. We then detail procedures for staining, FLEX tissue section collection, fixation, dissociation, and cell storage.