ATAC-seq of P2 mouse epithelial and non-epithelial cochlear cells

To identify gene regulatory regions in the cochlea, we FACS-sorted biological duplicates of epithelial cells (CD326+; including hair cells and supporting cells) and non-epithelial cells (CD326-; predominantly mesenchymal cells) from mouse cochlea at postnatal day 2 and performed ATAC-seq to identify open chromatin regions in each cell type. CD-1 timed-pregnant females were purchased from Charles River (Maryland). At postnatal day 2, the mice were euthanized and their temporal bone removed. Cochlear ducts from 20 mice were harvested, pooled and processed for Fluorescence-activated cell sorting (FACS). Briefly, single-cell suspensions were obtained and incubated with an APC-conjugated anti-CD326 (1:2000, BioLegend) to label epithelial cells. Cells were sorted into cochlear CD326 (+) cells (epithelial) and cochlear CD326 (-) cells (non-epithelial) by FACS using a BD FACS Aria II Cell Sorter (BD Biosciences) at the Flow Cytometry Facility, Center for Innovative Biomedical Resources (University of Maryland School of Medicine). 50,000 cells or 100,000 cells from each sample were further processed for ATAC-seq as described (Buenrostro et al. 2015. Curr. Prot. Mol. Med.) with the following modification: following the transposition reaction and purification step, a right side size selection (ratio 0.6) using SPRIselect (Beckman-Coulter, Indiana) was added before proceeding to the PCR amplification. This extra step resulted in the selection of DNA fragments between 150 bp to 700 bp. ATAC-seq data were generated from a total of four samples: two biological replicates from epithelial cells and two from non-epithelial cells.