Journal: Cell Reports Medicine

Article Title: Cell states and neighborhoods in distinct clinical stages of primary and metastatic esophageal adenocarcinoma

doi: 10.1016/j.xcrm.2025.102188

Figure Lengend Snippet: EAC primary and metastatic samples show a diverse landscape of TME and malignant cells in transcriptomic and epigenetic data (A) Schematic representation of the study workflow. Biopsies from 10 patients in our discovery cohort, including normal adjacent tissue (NAT), primary tissue, and metastatic samples, were subjected to single-nuclei RNA and ATAC sequencing using 10X Chromium technology. For a subset of these patients as well as three additional patients, matched primary and metastatic samples were profiled with 10X Visium and 10X Xenium spatial transcriptomics (ST) technologies. For single-nuclei data, cells were annotated by cell type and categorized into malignant and TME components. TME subtypes were linked to metastasis, with validation against an external pan-cancer fibroblast atlas. The malignant cell components underwent analysis using consensus non-negative matrix factorization (cNMF) to uncover malignant programs, which were further characterized for transcriptional and epigenetic heterogeneity at a single-cell and spatial level and candidate master transcription factors. External validation was performed in two single-cell validation cohorts, , and associations with clinical and molecular characteristics, as well as survival, were assessed in three bulk validation cohorts. , , (B) Uniform manifold approximation and projection (UMAP) representation of the full cohort in Harmony-corrected integrated transcriptomic data, with major cell type compartments labeled and cell counts indicated. (C) Proportion of major cell types in each sample based on transcriptomic data, with percentages for compartments representing over 5% of the total sample composition. (D) UMAP representation of the full cohort in Harmony-corrected integrated ATAC data, with cell type annotations transferred from the RNA annotations. “NA” denotes cells without paired associated RNA information. (E) Proportion of major cell types in each sample based on ATAC data, with percentages for compartments representing over 5% of the total sample composition.

Article Snippet: Next, the hardest coverslip was removed, and the sample deparaffinized according to the 10X Genomics Visium CytAssist Spatial Tissue Preparation guide ( CG000518 Rev C) and FFPE – deparaffinization and decrosslinking guide ( CG000520 Rev B).

Techniques: Sequencing, Biomarker Discovery, Labeling

Journal: Science Advances

Article Title: Spatially resolved analysis of microenvironmental gradient impact on cancer cell phenotypes

doi: 10.1126/sciadv.adn3448

Figure Lengend Snippet: ( A ) Schematic overview showing the collagen I/cell layer transfer to Visium slides. ( B ) Detailed process flow for the OCT-based treatment and transfer of collagen I/cell layer. ( C ) H&E-stained images of collagen I/cell layers that were either methanol fixed only (control) or methanol fixed and frozen in OCT to determine the effect of OCT-based treatment on cell morphology. ( D ) Effect of the OCT-based treatment on RNA integrity. On the left are bioanalyzer electropherograms of RNA extracted from collagen I/cell layers methanol fixed only (control) or methanol fixed and frozen in OCT. The x axis is the RNA length (nt), and the y axis shows the fluorescence units (FU), where the fluorescence measured correlates with RNA amount of a given size. The bar plot on the right summarizes the RNA integrity number (RIN) values of RNA extracts from each condition using three replicates per condition. Bar height shows the mean across all three technical replicates: Dots show individual replicates. ( E ) Detailed process flow for spatial RNA capture validation using the Visium TO slide. ( F ) On the top is a stitched bright-field image of a patterned collagen I/cell layer transferred onto a Visium TO slide then stained with H&E. On the bottom is a stitched fluorescence image of the same collagen I/cell layer after permeabilization, cDNA synthesis, and tissue removal. Dark areas on the border between adjacent tiles on the fluorescence image are due to stitching artifacts from the imaging system. MeOH, methanol.

Article Snippet: The TO slide was directly H&E-stained according to the Visium H&E Staining guide (10x Genomics, CG000160) but excluding the methanol fixation step and imaged on an EVOS M7000 system (Invitrogen) using the 10× objective and on the bright-field channel.

Techniques: Staining, Control, Fluorescence, Biomarker Discovery, cDNA Synthesis, Imaging

Journal: STAR Protocols

Article Title: Protocol for the generation of single-nuclei RNA-seq libraries and quantification of heterogeneous cell types activated during social interaction

doi: 10.1016/j.xpro.2024.103395

Figure Lengend Snippet:

Article Snippet: Note: We created our cDNA libraries following the 10x Genomicsuser guide “Chromium Next GEM Single Cell 3′ Reagent Kits v3.1” ( https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/chromium-single-cell-3-reagent-kits-user-guide-v-3-1-chemistry ) and sequenced these libraries on the Illumina NovaSeq.

Techniques: Recombinant, Saline, Isolation, Sequencing, Software, Transferring, Dissection