Single cell RNAseq of human macrophage polarization states
We employed the BD Rhapsody scRNAseq platform to identify human macrophage polarization states and their response to various stimulaiton conditions. Using a select panel of genes (BD Immune Response Panel Hs) we were able to identify unique transcriptomes for naive, IFNy, IFNb, IL-4, IL-10, and IL-13 polarized macrophages under unstimulated conditions and in response to 3 hours of acute stimulation with LPS, polyIC and TNF. Overall design: Human blood was obtained from a deidentified donor from the UCLA CFAR Centralized Laboratory Support Core in accordance with UCLA IRB 11-000443. Peripheral blood mononuclear cells were isolated from blood by Ficoll (Cytiva, 17-1440-03) density centrifugation. Monocytes were isolated using CD14+ bead selection (Milltenyi, 130-050-201), plated on 6-well tissue culture plates at a density of 5x105 cells/well, and cultured in RPMI (Gibco, 11875-093) supplemented with 10% ES fetal bovine serum (Gibco, 10439-024), penicillin-streptomycin (Corning, 30-002-Cl), L-glutamine (2mM; Corning, 25-005-CI) and 20ng/ml human macrophage colony-stimulating factor (M-CSF) (R&D systems, 216-MC-100). Media was replaced at day 4, and on 6 days from isolation, the cells were polarized for 24 hours with 100 U/mL IFN-ß (PBL Assay Science, 11415-1), 20 ng/mL IFN-? (Peprotech, 300-02), 50 ng/mL IL-10 (R&D, 217-IL), 40 ng/mL IL- 13 (R&D, 213-ILB), 20 ng/mL IL-4 (R&D, 204-IL), or left naïve. After 24 hours of polarization the cells were either left unstimulated or stimulated for 3 hours with LPS 1ng/ml (Sigma-Aldrich, L6529), TNF 10ng/ml (R&D, 210-TA-020), or polyIC 50ug/ml (Invivogen-TLRL-PIC). To collect the macrophages, the plate was washed with PBS, then incubated with 1mL warm 1mM EDTA for 15 minutes. Cells were lifted with a cell scraper, multiplexed using the BD Human Single-Cell Multiplexing Kit (BD, 633781), and loaded onto the cartridge following manufacturer's instructions (BD Rhapsody #210967). The LPS and TNF sitmulated samples were loaded on Cartridge 1 and the polyIC and unstimulated on Cartridge 2. Targeted mRNA amplification was performed utilizing the Immune Response Panel Hs primer panel (BD, 633750), with subsequent library preparation. The pooled library was sequenced with paired-end 100bp reads on an Illumina NovaSeq X Plus. After sequencing, the FASTQ files were aligned to the Immune Response Panel reference file, generating raw read counts per gene and per cell, which were then linked to cell samples of origin, with the final output being a cell by molecular matrix.