RNA-Seq of THP-1 macrophages uninfected
Total RNA extraction from five independent biological replicates of each infected and non-infected macrophages was performed 24 hours after infection using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) following manufacturer instructions. After, RNA samples were treated with DNase I (1 U/g of RNA) (Thermo Scientific, Lithuania, EU) at 37C for 1 h. The absence of RNA contamination was determined from the A260/A280 ratio using a spectrophotometer Nanodrop ND1000 (Thermo Scientific, USA). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer and a Pico Agilent RNA 6000 kit (Agilent Technologies, Santa Clara, CA, USA). rRNA depletion was performed using Ribo-Zero Human/rat/moRibo-Zero plus rRNA Depletion Kit (Illumina). Library preparation was performed using Truseq Stranded Total RNA LT Sample Prep Gold kit and RNAseq analysis was performed with NovaSeq 6000 S4 Reagent Kit (Illumina), according to the manufacturers instructions. RNA sequencing was performed with paired-ends (100bp) using the Illumina Novaseq 6000 Platform in Macrogen Inc. Service (Seoul, South Korea). Quality control of raw sequencing data was done using FastQC tool. Mapping to a human reference genome assembly (GRCh38) was done using bowtie2 (Langmead & Salzberg, 2012). Read counts from the resulting BAM alignment files were obtained with featureCounts using a GTF gene annotation from the Ensembl database (Howe et al., 2021; Y et al., 2014). The R/Bioconductor package edgeR was used to identify differentially expressed genes among the samples, after removing absent features (0 counts) (McCarthy et al., 2012). Genes with  adjusted p values less than 0.05 were identified as differentially expressed. For each time point comparison, gene set enrichment analysis was performed using the fgsea R package. Genes with Ensembl IDs were transformed into gene symbols by the biomaRt package (S et al., 2009)