RNA-seq analysis of in vitro polarised human monocyte-derived macrophage transcriptomes
A systematic study to model human macrophages polarised towards factors relevant to the atherosclerotic plaque environment. Our data revealed transcriptionally distinct phenotypes, each displaying significant enrichment of DEGs in atherosclerosis–related pathways. Monocytes isolated from healthy human donors were cultured in complete media: RPMI-1640 (Gibco), 10% (v/v) low–endotoxin heat–inactivated FBS (Biowest), 1% (v/v) L–glutamine (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Recombinant human (rh) M–CSF (100 ng/ml, Peprotech/Immunotools) was added to differentiate monocytes into monocyte–derived macrophages (MDMs) over 7 days. On day 7, the media was replaced with fresh complete media containing the following polarising agents: 20 ng/ml rhIFNγ (Peprotech/Immunotools) and 100 ng/ml TLR grade E. coli LPS (Enzo Life Sciences, R515), 20 ng/ml rhIL–4 (Peprotech/Immunotools/Miltenyi), 20 ng/ml rhIL–10 (Peprotech/Immunotools), 25 μg/ml oxPAPC (Invivogen) or 1 μM (7.8 μg/ml) rhCXCL4 (Peprotech/Immunotools/BioLegend), incubated for 24 h. Unpolarised macrophages (Mun) were used as internal baseline controls in each experiment. Polarised MDM from 8 separate donors were lysed in extraction buffer from the ARCTURUS® PicoPure® RNA Isolation Kit (ThermoFisher Scientific) and incubated at 42 °C for 30 min followed by centrifugation at 3,000 g, for 2 min. Supernatants were stored at – 80°C, until total RNA was extracted using the ARCTURUS® PicoPure® RNA Isolation Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Total RNA integrity was assessed using an Agilent Bioanalyzer with RNA Integrity Number (RIN) ≥ 8.5. cDNA libraries were prepared using 2 ng of total RNA and 1 μl of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion) using SMARTSeq v2 protocol. The length distribution of the cDNA libraries was determined using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip. All samples were subjected to an indexed pair–end sequencing run of 2 × 51 cycles on Illumina HiSeq 2000 (16 samples/lane). RNA-Seq data in FASTQ files were obtained and mapped using STAR against build 38 of the human genome. The number of reads per gene was counted using feature Counts (part of Subread package) using annotations from GENCODE (v 24). Log2RPKM values were computed using edgeR in R (v 3.1.2) and used for Principle Component Analysis (PCA). Pairwise differential gene expression analyses for each of the cell types were performed using edgeR with unpolarised macrophage samples as the reference. Differentially expressed genes (DEGs) were identified using False Discovery Rate (FDR) of < 5% (Benjamini and Hochberg method) and fold change |log2FC| >log2(1.5) when compared to unpolarised macrophages. The data table contains the mean values from 8 donors. Raw sequencing information is not available to comply with ethical approval.