GSM9292445: E16.5 Mouse Cochlea, SCR240, Ebf1-cKO, ATAC; Mus musculus; ATAC-seq
After collecting E14.5 or E16.5 embryos from Ebf1fl/fl females timed-mated with Slc26a9P2A-Cre Ebf1fl/fl males, tail snips were gathered for Slc26a9P2A-Cre qPCR genotyping, and the temporal bones were removed. The cochlear ducts were then dissected in 1% 1 M HEPES diluted in HBSS and pooled according to genotype in cryogenic tubes. Next, the saline solution was removed, and the cochlear ducts were snap frozen and stored in liquid nitrogen. Once E14.5 and E16.5 samples with at least six Slc26a9P2A-Cre Ebf1fl/fl and six Ebf1fl/fl cochlear ducts had been obtained, the samples were thawed on ice, and the nuclei were isolated using a modified version of the protocol for the Chromium Nuclei Isolation Kit (10x Genomics 1000493) that omitted steps involving vortexing or use of a pestle or column. Briefly, the samples were lysed and then filtered using tubes with cell strainer caps (FALCON 352235) before being spun down at 500 RCF for 3 min at 4°C. The nuclei pellets were then washed, centrifuged at 500 RCF for 10 min at 4°C, and resuspended. Following nuclei isolation and purification, samples were centrifuged at 300 RCF for 5 min at 4°C and resuspended in HBSS to reach a concentration of 5000 cells/µL. Library construction was done with a 9,000 targeted nuclei recovery using the Chromium Next Cell Multiome ATAC + Gene Expression Reagent Bundle (10x Genomics 10000285) following the manufacturer's protocol. Briefly, nuclei were encapsulated in a gel matrix and uniquely barcoded using the 10x Chromium Controller (Chip J, 10x Genomics 1000230). Libraries were then sequenced using Illumina NovaSeq.