design: A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning - scRNA-seq
library_construction_protocol: In this study, human embryonic cochleae were collected from medically aborted embryos ranging from 10 to 17 PCW. The study protocol was reviewed and approved by the Ethics Committee of the Eye, Ear, Nose and Throat Hospital of Fudan University, China (Approval No.: 2022166). None of the samples utilized in this study exhibited known genetic abnormalities. Fetuses with severe pregnancy-related complications or congenital developmental abnormalities were excluded. Prior to the procedure, informed consent was obtained from all participating pregnant women after comprehensive counseling, authorizing the use of fetal tissue for research purposes. Fetuses with severe pregnancy lesions and abnormal fetal congenital development were excluded. All patient and sample information was numbered and collected anonymously. For fetal cochlear explant culture, temporal bone was disassociated from aborted fetus. The temporal bone was washed with 1.5 µg/mL ampicillin in ice-cold PBS. The cochleae were isolated with sterile forceps under a stereoscopic microscope. For scRNA-seq, cochlear tissues were dissociated into single-cell suspensions by mincing and trypsin digestion at 37 °C for 20 min. After neutralization and filtration through a 40 μm strainer, cells were treated with RBC lysis buffer, washed, and resuspended in MEM supplemented with 1% BSA and RNase inhibitor. Cell concentration and viability were assessed using a LUNA-FL™ automated cell counter with LIVE/DEAD staining. For scRNA-seq, single-cell suspensions with viability exceeding 80% were used for scRNA-seq library preparation. Libraries were constructed according to the manufacturer's protocol using the Chromium Controller instrument and the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1. Library preparation was carried out according to the manufacturer's protocol.