title: GSM9163262: scRNA-seq of mouse cochlea transduced with AAV1-1.0kb-mMyo15-OTOF-v5, biol. rep. 2; Mus musculus; RNA-Seq
library_construction_protocol: For scATAC-seq, 1X lysis and wash buffers were prepared according to 10x Genomics' Demonstrated Protocol CG000212. One live cochlea was placed into a 1.5 mL Eppendorf tube on ice and 100 uL 1X lysis buffer was added. The cochlea was broken up with spring scissors and then incubated in the lysis buffer for either 5 min or 10 min. 1 mL chilled wash buffer was added to the lysed cells and the nuclei suspension was filtered with a 20 µm pluristrainer (Pluriselect-USA, El Cajon, CA, USA) to remove debris. For scRNA-seq, micro-dissected tissue from each mouse was pooled in a solution of DMEM/F-12 medium containing 200 units/mL collagenase IV (Thermo Fisher Scientific, Waltham, MA) and 10 kunitz/mL DNase I (STEMCELL Technologies, Cambridge, MA) and incubated for 30 min at 37 °C to digest extracellular matrix. The collagenase IV solution was then replaced with EBSS containing 20 units of papain (Worthington Biochemical, Lakewood, NJ, USA), 1 mM L-cysteine, 0.5 mM EDTA, 15 mM HEPES, and 15 kunitz/mL DNase I and incubated for an additional 30 min at 37 °C, triturating with a 1000 µL pipette every 10 min to generate a single cell suspension. An equal volume of DMEM/F-12 medium containing 20% ovomucoid protease inhibitor (Worthington Biochemical, Lakewood, NJ, USA) was added, and the dissociated cells were passed through a 40 µm filter (PluriSelect-USA, El Cajon, CA) to remove large debris. The cells were pelleted at 350g for 5 min, washed twice, and then pelleted and resuspended in DMEM/F-12 containing 0.08 % BSA. Finally, a 10 µL sample of the cell suspension was counted on a Luna-FL automated counter using AO/PI cell viability staining (Logos Biosystems, Annandale, VA, USA). For scATAC-seq, the nuclei were centrifuged at 500 xg for 5 minutes, resuspended in diluted nuclei buffer, and counted with the Luna-FL. Transposition, nuclei capture, and library preparation were performed according to the 10x Genomics protocol using ATAC v1 reagents. For scRNA-seq, cells were captured, lysed, and primed for reverse transcription (RT) using the high-throughput, droplet microfluidics Gemcode platform (10x Genomics, Pleasanton, CA, USA) with v3 chemistry.