METTL14 regulates chondrogenesis through the GDF5–RUNX–extracellular matrix gene axis during limb development [m6A-seq]
METTL14 forms a protein complex that induces m6A methylation on RNA but its roles in mouse limb development remains elusive. We knocked out Mettl14 in a limb bud-specific manner with the Prx1 promoter-driven Cre. The resulting Prx1-cre;Mettl14 flox/flox (conditional KO or cKO) mice showed shorter limbs with disrupted bone and cartilage development compared with the heterozygous Prx1-cre;Mettl14 wild type/flox (cHet) control mice. To understand the molecular basis for the abnormalities, we applied total RNA from day 12.5 (E12.5) embryonic limb buds to m6A-seq. Overall design: RNA prepared from cKO and cHet limb buds on E12.5 was applied to m6A-seq as follows. First, a small fraction of the RNA was analyzed with RNA-seq as input. Second, m6A-modified RNA was immunoprecipitated with anti-m6A antibody from the remaining RNA and applied to the second RNA-seq as IP (immunoprecipitated). The sequencing results with a pair of input and IP for six groups (biological triplicates for cKO and cHet each) are uploaded.