RNA-seq of pirmary human monocytes stimulated with C-reactive protein in the presence or absence of PLGA and PEG-PLGA nanoparticles
RNA-seq of pirmary human monocytes stimulated with C-reactive protein in the presence or absence of PLGA and PEG-PLGA nanoparticles
Peripheral blood mononuclear cells (PBMCs) were isolated from pooled buffy coats of healthy human donors using density gradient centrifugation on Histopaque. Monocytes were isolated via magnetic-activated cell sorting (MACS) using a Pan Monocyte Isolation Kit (Miltenyi Biotec) based on negative selection, leaving monocytes untouched. Cell purity was verified by flow cytometry (CD45, CD14). Isolated monocytes were seeded at 5×10⁶ cells/mL in RPMI 1640 with 10% FBS and 1% Pen/Strep, rested 2 h at 37 °C, then stimulated with nanoparticles ± CRP for 4 h. Monocytes were stimulated for 4 hours at 37 °C with combinations of recombinant human C-reactive protein (CRP; 90 µg/mL), and polymeric nanoparticles (PLGA, PEG-PLGA 2/11.5 kDa, PEG-PLGA 5/25 kDa; 85 µg per million cells). Nanoparticles pre-adsorbed with CRP were incubated for 45 min at 37 °C before stimulation. Unstimulated and no-nanoparticle controls were included. Following stimulation, cells were lysed directly in wells with 400 µL TRI Reagent (Zymo Research). RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer's instructions, including in-column DNase I treatment. RNA quality was confirmed by Agilent TapeStation (RIN ≥ 6) and quantified with NanoDrop Library preparation was performed by Single Cell Discoveries (Utrecht, NL) using an adapted CEL-Seq protocol (Hashimshony et al., 2012; Simmini et al., 2014). RNA samples were normalized and barcoded during reverse transcription, pooled after second-strand synthesis, and amplified by in vitro transcription. Amplified RNA served as input for Illumina-compatible library construction using TruSeq small RNA primers. Library quality was confirmed by Agilent Bioanalyzer.