METTL14 regulates chondrogenesis through the GDF5–RUNX–extracellular matrix gene axis during limb development [RNA-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 analyzed polysome RNA prepared from day 12.5 (E12.5) embryonic limb buds with RNA-seq. Cytoplasmic fractions were prepared from cKO and cHet limb buds on E12.5 and loaded to sucrose gradient ultracentrifugation to purifiy polysome fractions. Total RNA prepared from the cytoplasmic and polysome fractions were labeled as input and polysome RNA, respectively and applied to RNA-seq.