Quantitation of stereocilia dimensions in transduction and Myo15a mutants

Transduction mutant length and width data.xlsx contains data for Tmie KO and Tmc DKO mice at P7.5 and P21.5.

sh2 length and width data.xlsx contains data for Myo15a^sh2 mice at P8.5.

For analyzing length and width of apical IHC stereocilia of Tmie, Tmc1;Tmc2 and Myo15a^sh2 mutant mice at P7.5 and P21.5, z-stack images of phalloidin-stained IHC stereocilia from cochleas of mutant mice were obtained using identical image acquisition parameters. Images were acquired on a ZEISS Elyra PS.1/LSM 710 Airyscan combination setup, using a 63x 1.4 NA Plan-Apochromat or a 100x 1.46 NA alpha Plan-Apochromat lens, integrated under ZEN 2.1 software. Raw data processing for Airyscan-acquired images was performed using manufacturer-implemented automated settings. At least 2-4 cochleas were used for each genotype. Images containing hair bundles that were upright were selected for generating x-z reslices of stereocilia with the ‘Reslice’ function in Fiji. Reslices were chosen with views of stereocilia rows 1 and 2; we chose central stereocilia in most bundles. Stereocilia lengths were measured individually in Fiji by manually drawing a vertical line from the top of a stereocilium to the point of insertion in the cuticular plate. For measuring stereocilia widths, horizontal lines were manually drawn at 50% of the bundle height for both rows 1 and 2.