RNA-Seq Analysis of Macrophage Transcriptome

For RNA-Seq, macrophages were washed twice with cold PBS and lysed in TRIzol reagent (Thermo). RNA was isolated from the aqueous phase using RNeasy kits (Qiagen). RNA with RIN > 8 was subjected to poly-dT pulldown using magnetic beads (NEB) before preparation for RNA-Seq using RNA Ultra kits (NEB). Libraries were sequenced on a NextSeq 500 (Illumina) and reads were aligned to the mm10 transcriptome using HISAT2 ^{29 (link)} after adaptor trimming using cutadapt ³⁰ . Reads counts per gene for RefSeq genes were computed using featureCounts ^{31 (link)}. Counts were normalized to reads per kilobase per million (RPKM) and processed for pairwise differential expression analysis of selected conditions using DESeq2 ^{32 (link)} with an False Discovery Rate (FDR)-adjusted p-value cutoff of 0.05. Gene Ontology analysis was performed using the PANTHER database ^{33 (link)} and sequence motif analysis was performed using HOMER ^{34 (link)}.