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To summarize, total RNA was isolated from liver macrophage samples utilizing TRIzol reagent (TaKaRa, Japan). Sequencing libraries were constructed from low-input RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, Japan). The process involved cDNA synthesis, followed by purification and size selection (150-300 bp). Sequencing-ready libraries were then prepared via PCR-mediated addition of i5 and i7 adapters, with quality control (size distribution and concentration) assessed on an Agilent 2100 Bioanalyzer, prior to being sequenced on an Illumina platform under a 2x150bp paired-end protocol. For bioinformatics analysis, raw reads were first processed with Cutadapt (version 1.9) to remove adapter contamination. After filtering out low-quality and undetermined bases, the clean reads were aligned to the reference genome (Mus musculus, Ensembl release 96) using HISAT2 (version 2.0.4). Transcript assembly for each sample was performed with StringTie (version 1.3.4d), and expression levels were quantified in FPKM. Differential expression analysis of mRNAs was conducted using edgeR, applying thresholds of fold change > 2 and p-value < 0.05. Finally, the differentially expressed mRNAs were functionally annotated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. |
RNA-Seq |
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PCR |
PAIRED
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