title: GSM6883298: Cochlea_wildtype_P8_multiome_ATAC; Mus musculus; ATAC-seq
library_construction_protocol: Cochlear tissue was dissected from P1 and P8 mice and enzymatically dissociated with a cocktail of 400µl of 0.25% Trypsin, 50µl of 1 mg/ml Dispase, and 50µl of 1 mg/ml collagenase for 20 minutes at 37°C. After incubation, the digested tissue was titurated 100 times using a small-bore 200µl pipette and centrifuged at 500x g for 5 minutes at 4°C. After centrifugation, the sample was processed according to the 10x Genomics protocol “Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing” (CG000375, Rev B). Briefly, the supernatant was decanted, and the cell pellet was resuspended in 1ml of NP-40 Lysis Buffer and incubated for 5 min on ice. Nuclei were filtered through a 40 micron cell strainer, and centrifuged at 500x g for 5 minutes at 4°C. The supernatant was decanted, and nuclei were resuspended in 1ml of PBS + 2% BSA and incubated for 5 min at 4C. Nuclei were centrifuged at 500x g for 5 minutes at 4°C, resuspended in 100ul of 0.1x Lysis Buffer, and mixed with a pipet. Nuclei were incubated for 2 minutes on ice, followed by the addition of 1ml Wash Buffer, mixed and centrifuged at 500x g for 5 minutes at 4°C, and resuspended in the appropriate volume of Diluted Nuclei Buffer for input into the Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics). For the P1 cochlea, 6,065 nuclei were loaded. For the P8 cochlea, 9,645 nuclei were loaded. scMultiome ATAC + Gene Expression protocol (10x Genomics).