Forming pinna prominences from E14.5 embryos (both wild type and mEC1dup/dup) were microdissected, washed with cold 1× PBS, and homogenized using a Tissue-Tearor. RNA extraction was performed according to the manufacturer's protocol (RNeasy Mini Kit, Qiagen, 74104). A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair,A-tailing and adapter added were implemented. The aimed products were retrieved and PCR was performed, then the library was completed.