A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning- scRNA-seq-2
A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning- scRNA-seq-2
Human cochlear organoids were generated from human pluripotent stem cell–based differentiation and served as the biological samples for this study. Organoids were maintained in CHIR99021-containing proliferative medium (EFI) and expanded for 10 days. Subsequently, organoids were subjected to adeno-associated virus (AAV)–mediated gene activation and cultured for an additional 7 days under differentiation conditions prior to sample collection.  At the designated collection time point, cochlear organoids were harvested and immediately transferred to ice-cold oxygenated artificial tissue preservation solution.  For scRNA-seq, harvested organoids 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.