title: GSM8979935: Wild_type_MacrophagesM2_S6; Mus musculus; miRNA-Seq
library_construction_protocol: Long bones were extracted from the hind limbs of mice under sterile conditions. Bone marrow aspirate was obtained using the protocol previously described (Marinescu et al, 2021). Briefly, medullary channels were flushed with 5 ml of medium (high glucose DMEM supplemented with 10% foetal fetal bovine serum) using a 25-G needle connected to a 5-ml syringe. Subsequently, the single-cell suspension obtained by passing the bone marrow aspirate through needles of decreasing sizes (18G, 21G, 23G, 25G) was resuspended to a concentration of 106 cells/ml and used for further experiments. Monocytes were obtained using EasySep™ Mouse Monocyte Isolation Kit (STEMCELL Technologies, #19761) following the manufacturer's instructions.  Briefly, around 60x106 bone marrow aspirate was resuspended in 1 ml of EasySep buffer (STEMCELL Technologies, #20144) and incubated with an antibody cocktail targeting non-monocyte cells, including erythrocytes, lymphocytes, neutrophils, and dendritic cells. Afterward, EasySep™ Dextran RapidSpheres™ were added to the cell suspension to bind the antibody-labelled cells. The suspension was then placed into an EasySep™ magnet, which selectively retains the magnetically labelled cells while allowing the untouched monocytes to be transferred into a new tube. The isolated monocytes were washed, counted, and either used for flow cytometry analysis or cultured in vitro to differentiate into macrophages for further experiments.                                                                                                     Isolated monocytes were seeded in culture-treated plates at a density of 40,000 cells/cm² in a differentiation medium i.e., high glucose DMEM supplemented with 20% FBS and 30% L929 conditioned medium (L929-CM), as the source of M-CSF. A medium refresh was done after three days of culture. After seven days of differentiation, the cells (M0 macrophages) were polarized towards M1 or M2 phenotypes, by incubating with 100 ng/ml LPS and 20 ng/ml IFN-γ for M1 polarization, or with 20 ng/ml IL-4 for M2 polarization, for the next three days. Cells maintained cultured in the differentiation medium without cytokines served as control. Total RNA was isolated from cells using the standard protocol of TRIzol reagent (Thermo Fisher Scientific, #15596026). One µg of total RNA was used for reverse transcription, using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA#4387406). The RT-PCR was performed on the ViiA™ 7 Real-Time PCR System using SYBR™ Select Master Mix (ThermoFisher ScientificApplied Biosystems, #4472918). Relative expression level was determined using the comparative CT method, with RPL32 used for normalization. miR-210 expression levels were measured using TaqMan™ MicroRNA Assays (ThermoFisher Scientific, #A25576) in combination with the TaqMan™ MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, #4366596). The expression of U6 snRNA was used as an endogenous control for normalization. Details are provided in the materials and methods section