Chromium single cell 3 gene expression system

Single cell whole transcriptome profiling of peritoneal cells was performed using the Chromium Single-Cell 3′ Gene Expression system from 10X Genomics. Single cells were resuspended in PBS buffer at 10⁶ cells/mL and loaded onto Chromium chips. Single-cell capturing, barcoding, and cDNA library preparation were performed using the Chromium Single-Cell 3′ Library & Gel Bead Kit v2 (10X Genomics, Pleasanton, CA, USA) per manufacturer protocol. The final sequencing libraries were checked for quality on Agilent 4200 Tapestation System (Santa Clara, CA, USA) and quantified by fluorometry staining (QuBit) assay. Libraries were sequenced on a HiSeq4000 (Illumina, San Diego, CA, USA) at a depth of ~50,000 reads per cell. The scRNA-seq was analyzed using standard Seurat v3 [64 (link)] integration workflow. Top 2000 variated features were selected to find anchors between pairs of datasets. The integrated dataset was dimension-reduced and visualized using the Uniform Manifold Approximation and Projection (UMAP) with top 30 principal components. All cells are clustered using the original Louvain algorithm. Dot plots, violin plots, and heatmaps were generated using the R Seurat package (version 5). The scRNA-seq data are available at Gene Expression Omnibus (GEO), GSE228598.

Ascites or peritoneal washing samples were prospectively collected from 28 patients undergoing diagnostic laparoscopy or paracentesis (diagnostic or therapeutic) for biopsy-proven gastric adenocarcinoma (stage I–IV) with or without peritoneal metastases. Inclusion criteria were an age greater than 18 years old and known diagnosis of gastric cancer. The exclusion criterion was the inability to give consent. Gross peritoneal disease was neither considered inclusion nor exclusion criteria. During diagnostic laparoscopy, peritoneal specimens were collected after lavage with ~1000 mL of normal saline with laparoscopic access. For GC patients with peritoneal carcinomatosis and malignant ascites, up to 5 L of the discarded ascites collected during a paracentesis was obtained. The first 80 mL–160 mL of the specimens was sent to pathology for routine cytologic evaluation. The remaining samples were immediately placed on wet ice for single-cell processing. The fluid was spun down, and the cell pellet was collected and resuspended in phosphate-buffered saline (PBS, pH 7.4). The cell count was obtained and prepared for single-cell RNA sequencing (scRNA-seq) using the Chromium Single-Cell 3′ Gene Expression System from 10X Genomics (Pleasanton, CA, USA).