GSM5236060: Cytoplasm_SmallRNA_THP1_monocyte_0h; Homo sapiens; miRNA-Seq
Nuclear and cytoplasmic THP-1 fractions were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher) according to instructions, with additional nuclear washes prior to nuclear extraction. Fractionation purities were assessed by b-tubulin and Lamin B2 contamination. THP-1 monocyte and THP-1 macrophage exosomes were isolated by differential centrifugation (10 min x 300 g, 10 min x 2000 g, 30 min x 10,000 g, 70min x 100,000 g all at 4°C), washed and resuspended in PBS for analysis. Exosome particle size distributions were assessed by nanoparticle tracking analysis (NanoSight). Small RNA was immediately isolated from subcellular fractionation experiments using the mirVana miRNA isolation kit according to instructions (AM1560, Invitrogen). Small RNA libraries were generated using the NEBnext small RNA library preparation kit according to instructions (E7580S, New England BioLabs)