A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning- scRNA-seq-2

Human cochlear organoids were derived from human pluripotent stem cell–based differentiation and maintained in CHIR99021-containing proliferative medium (EFI). Organoids were cultured under proliferative conditions for 10 days, after which they were transduced with 3 types of adeno-associated virus (AAV). Following viral transduction, organoids were switched to differentiation conditions and cultured for an additional 7 days. At the designated time point, cochlear organoids were harvested and immediately transferred to ice-cold oxygenated artificial tissue preservation solution. Under microscopic guidance, organoids were gently dissociated to obtain single-cell suspensions. Enzymatic digestion was performed using 0.125% trypsin at 37°C for 20 minutes, followed by gentle mechanical trituration. The resulting cell suspension was filtered through a 40 µm cell strainer and subjected to red blood cell lysis where necessary. Cell viability was assessed using a LUNA-FL automated cell counter with LIVE/DEAD staining, and samples with viability exceeding 80% were processed for downstream analysis. Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3' Reagent Kits v3.1 (10x Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 platform with 150 bp paired-end reads.