Single-cell RNA-seq analysis of mouse liver

As described previously, mouse livers were digested to obtain single-cell suspensions A [20 (link)]. Dissociated single cells were stained with AO/PI for viability assessment using a Countstar Fluorescence Cell Analyzer. The scRNA-seq libraries were generated using the 10× Genomics Chromium Controller Instrument and Chromium Single Cell 3’V3.1 Reagent Kits (10× Genomics, Pleasanton, CA, USA) according to the recommendations of the manufacturer. scRNA-seq data analysis, including cell communication analysis, quantitative set analysis of gene expression (QuSAGE) analysis, and pathway analysis were performed by NovelBio Bio-Pharm Technology Co., Ltd., using the NovelBrain Cloud Analysis Platform as previous procedures [29 (link)–32 (link)]. To characterize the relative activation of a given gene set, such as pathway activation, we performed QuSAGE analysis, in which gene sets, including ferroptosis, described previously, were added. Pathway analysis was used to identify the significant pathways of the marker genes and differentially expressed genes according to the kyoto encyclopedia of genes and genomes (KEGG) database. Fisher’s exact test was used to select significant pathways, and the threshold of significance was defined by the P-value and false discovery rate (FDR).