title: GSM9564255: Spatial transcriptomics of bone metastatic tissue section 1; Mus musculus; OTHER
library_construction_protocol: For single-cell RNA sequencing, bone metastatic tissues were carefully dissected and processed into single-cell suspensions following previously established protocols as described in “Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization” (Xu et al., bioRxiv, 2024). Briefly, tissues were mechanically minced and enzymatically dissociated under controlled conditions to preserve cell viability. The resulting cell suspensions were filtered to remove debris and cell aggregates, washed, and counted prior to downstream single-cell library preparation, as previously reported  2024.05.07.593016v1.full .  For spatial transcriptomics, bone tissues were freshly harvested and processed according to the 10x Genomics Visium Spatial Gene Expression demonstrated tissue preparation protocol (CG000240, Rev A). When necessary, bone tissues were decalcified using an RNA-preserving EDTA-based protocol prior to embedding. Samples were embedded in optimal cutting temperature (OCT) compound, snap-frozen, cryosectioned, and mounted onto 10x Genomics Visium Spatial Gene Expression Slides. Tissue handling, cryosectioning, and slide preparation were performed following the Visium Spatial Protocols – Tissue Preparation Guide to preserve tissue morphology and RNA integrity. All procedures were carried out under RNase-free conditions prior to library preparation according to the manufacturer's instructions Single-cell RNA-seq libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Gene Expression platform following the manufacturer's protocol. Briefly, single cells were encapsulated into gel bead-in-emulsions (GEMs), followed by reverse transcription, cDNA amplification, and library construction. For the details, please refer to “Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization” (Xu et al., bioRxiv, 2024)  Spatial transcriptomics libraries were generated using the 10x Genomics Visium Spatial Gene Expression protocol (CG000240). Tissue sections were fixed, stained, imaged, permeabilized, and reverse-transcribed on-slide. Spatially barcoded cDNA was collected, amplified, and converted into sequencing libraries following the manufacturer's instructions.