Enhanced crosslinking and immunoprecipitation analysis of human fetal ß-cells
N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA, and plays important roles in human embryonic stem cell pluripotency, maintenance, and differentiation. However, the role of m6A and the precise mechanisms involved during the development of ß-cells are unexplored. Here, we performed enhanced crosslinking and immunoprecipitation (eCLIP) assays to identify m6A sites regulated by METTL14 in EndoC-ßh1 human fetal ß-cells. Overall design: EndoC-ßH1 cell lines were obtained from Univercell-Biosolutions (France). Culture plates were coated with DMEM (glucose 4.5 g L-1; Gibco) containing fibronectin (2 µg mL-1; Gibco), and extracellular matrix (1% vol vol-1; Sigma) for at least an hour in 5% CO2 at 37°C. EndoC-ßH1 cells were grown on coated 6-well plates containing DMEM (glucose 1 g L-1), BSA fraction V (2% wt vol-1) (Roche), 2-mercaptoethanol (50 µM; Sigma), nicotinamide (10 mM; Sigma), transferrin (5.5 µg mL-1; Sigma), and sodium selenite (6.7 ng mL-1; Sigma). The eCLIP assays were performed by Eclipse Bioinnovations using UV-crosslinked EndoC-ßH1 cells following the protocol detailed in (Van Nostrand et al. Nat Methods. 2016) using the anti-METTL14 antibody A305-847A (Bethyl Laboratories).