Molar Tooth Germ Transcriptomics

SD rats with a tooth in the differentiation stage at postnatal day 1.5 (P1.5) and secretory stage at postnatal day 3.5 (P3.5) were treated with inhalation anesthesia. We chose a 30% volume displacement rate per minute for CO₂ euthanasia of rodents and assisted by cervical dislocation. After euthanasia, we separate the head along the upper neck and then dissected the mandible along the corner of the mouth. The location of the mandibular molar tooth embryo was found at the mandibular ascending ramus below the coracoid process. Finally, tooth germs were dissected with ophthalmic tweezers under a stereo microscope (Olympus SZ61, Tokyo, Japan) and photographed with a microscope camera (MSHOT, Guangzhou, China) (Figures 1(a) and 1(b)). Samples were put in solidified carbon dioxide and immediately sent out for high-throughput sequencing (Genesky Biotechnologies Inc, Shanghai, China). We compared gene expression from the secretory stage to the differentiation stage, and each group was prepared as three individual samples. In addition, we prepared three additional rats per group for follow-up validation experiments. To ensure their development stage, pups outside the expected size range were excluded.