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/).