title: GSM5824447: Donor 1 MCSF; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from peripheral blood using a Ficoll-Paque (GE Healthcare) density gradient. Monocytes were isolated from PBMCs by adherence for 2 hours with RPMI supplemented with 1% fetal calf serum (FCS) or via positive selection with CD14 microbead (Miltenyi Biotec, cat# 130-050-201). Monocytes were seeded at 500,000 cells/well in a 24-well plate and differentiated in M-CSF (50 ng/ml) (R&D Systems, cat. # 216MC025/CF) for 5 day in RPMI with 10% FCS at 37°C. In vitro 5-day M-CSF derived macrophages were then stimulated with either M-CSF (50 ng/ml) (R&D Systems, cat. # 216MC025/) and/or squalene (1 mM) (Thermofisher Scientific, cat.# S3626-100ML). These macrophages were separated from culture plates with trypsin-EDTA and filtered through a 70 μm cell strainer and washed with 1ml of RPMI (Gibco, cat. # 11875093). The macrophages were centrifuged at 300g for 10 mins at 4°C. After discarding the supernatant, the cells were suspended in 1mL of RPMI and counted. Live cells were counted using 0.4% trypan blue (Gibco, cat. # 15250061). If the cell viability was above 90%, we loaded isolated cells onto the Chromium instrument (10X Genomics) as described above. Single cell RNA-seq sequencing, alignment, and analyses are described above. Isolated cells were loaded into the Chromium instrument (10X Genomics) according to manufacturer's protocol, sequenced with NovaSeqS2, and analyzed using Seurat.