title: A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning - sn-Multiome
design: A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning - sn-Multiome
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. Single-nucleus RNA and ATAC sequencing (snMultiome) was performed on cochlear tissues from three human fetuses (11, 14, and 16 PCW) using the 10x Genomics Chromium Multiome platform. Nuclei were isolated by mincing snap-frozen samples in ice-cold lysis buffer, followed by filtration, centrifugation, and mild permeabilization. After quality assessment via trypan blue staining, intact nuclei were resuspended in dilution buffer, quantified, and adjusted to 4,000–8,000 nuclei/μL for subsequent library preparation and sequencing. For snMultiome-seq, libraries were prepared and loaded following the manufacturer's instructions of the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle. Chip J (10x Genomics) was used for droplet generation. For the gene expression library, Dual Index Kit TT Set A (10x Genomics) was employed, while the ATAC library was constructed using Single Index Kit NT Set A (10x Genomics). 