title: GSM9003635: Macrophages IFN; R3; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood-derived monocytes were isolated from cryopreserved peripheral blood mononuclear cells obtained from 3 different healthy donors (Stemcell) using pan-monocyte isolation kit (Miltenyi) with autoMACS Neo cell separator (Miltenyi) according to the manufacturer's instructions. Cells were cultured in ImmunoCult SF Macrophage medium (Stemcell) containing 50 ng/mL of recombinant human M-CSF (Stemcell) in temperature-responsive polymer-treated UpCell plates (Thermo Fisher) for 4 days, followed by either supplementation with 50 ng/mL of recombinant human IFN-gamma (Stemcell) or replacement with supernatant of exhausted T-cells (Tex sup) for an additional 2 days. For Tex sup production, a serial killing assay was conducted using healthy donor-derived CAR T cells and HT cell line, starting at a 1:1 ratio in IMDM (Gibco) supplemented with 10% FBS (Gibco). HT cells were added every 2 days along with medium exchange. The supernatant was collected on day 10 and then cryopreserved until use. Tex sup and ImmunoCult SF macrophage medium were mixed at a 1:1 ratio and supplemented with 50 ng/mL rhM-CSF. RNA was extracted using RNeasy micro kit (QIAGEN) according to the manufacturer's protocol after washing the cells with PBS, followed by library preparation for RNAseq. Illumina NovaSeq 6000 platform, using the PE100 S4 flow cell for paired-end sequencing with a 200-cycle v1.5 kit.