Whole-transcriptome analysis with RNA-seq
Total RNA was extracted from the livers of mice using the RNeasy Mini Kit (Qiagen Inc., Hilden, Germany), libraries were prepared as previously described (Kohno et al., 2020; Muto et al., 2023), and the quality and concentration of the libraries were evaluated using Agilent 2200 TapeStation (Agilent tech. Inc., D1000). The libraries mixed to equal molecular concentrations were sequenced on an Illumina NextSeq500 DNA sequencer with a 75-bp paired-end cycle sequencing kit (Illumina Inc., San Diego, CA). The data were then trimmed and mapped to the mouse reference genome GRCm38 release-95 using the CLC Genomics Workbench software (ver.12.0.3; Qiagen Inc., RRID:SCR_011853). The mapped read counts were normalized to transcripts per million (TPM) and converted to log2 after adding 1. Differentially expressed genes with p-value < 0.05 and fold change > |1.2| were used for Ingenuity Pathway Analysis (Qiagen Inc., RRID:SCR_008653), which was performed to analyse the detected genes. A Venn diagram was created using the Calculate and draw custom venn diagrams software (http://bioinformatics.psb.ugent.be/webtools/Venn/). All heat maps were plotted as z-scores calculated from the TPM of each gene in the RNA-seq data using GraphPad Prism software (version 9.0; GraphPad Software, RRID:SCR_002798). The marker genes in scRNA-seq were referred against the mouse NAFLD myeloid cell atlas dataset (Guilliams et al., 2022). The genes listed in the referenced data were used to confirm specificity in monocyte and macrophage clusters among mouse NAFLD liver cells using public databases (https://www.livercellatlas.org/), and genes with higher specificity were selected. UMAP visualization of mouse NAFLD liver cells or NAFLD myeloid cells was performed using public datasets (https://www.livercellatlas.org/).