GSM9249200: 181121_cochlea_2D; Mus musculus; ATAC-seq
Neonatal (P2, n=3; P5, n=3; P8, n=4) CD1 mice were sacrificed and cochleas were dissected bilaterally as described above. All tissue from each animal was combined and dounced 10 times in 5 ml of HB buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl 2 , 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, DTT 1 mM [Sigma], one-half Roche proteinase inhibitor tablet) in a 7-ml Dounce homogenizer with tight pestle, then supplemented with IGEPAL CA-630 to final concentration of 0.3%, and finally dounced an additional five times with the tight pestle. Nuclei were filtered through a 40-μm strainer and gently combined 1:1 with 50% iodixanol. Nuclei were then underlaid with a gradient of 1 ml 40% iodixanol and 1 ml 30% iodixanol and centrifuged at 10,000g for 19 minutes using the no brake setting. Purified nuclei (0.8 ml) were then collected from the 30/40% interface. A 20-μl aliquot was taken for visual inspection and cell counting, with typical yield of ~100,000-200,000 cochlear nuclei per animal. ATAC-seq (assay for transposase accessible chromatin with sequencing) was performed using the OMNI-ATAC protocol 58 immediately following the nuclear isolation steps described above. Nuclei (50,000) were pelleted at 500 g for 10 minutes at 4°C in a fixed angle centrifuge and resuspended in 50 μl transposition buffer (prepared from Illumina kit components TD buffer and 100 nM transposase, as well as 0.01% digitonin, 0.1% Tween-20) via careful pipetting. Transposition occurred at 37°C in a thermomixer at 1000 rpm for 30 minutes before resuspension in 250 μl of DNA binding buffer (Zymo DNA Clean and Concentrator Kit) and reaction cleanup per manufacture instructions, with final elution volume of 21 μl in elution buffer. Samples were amplified following NEBNext High-Fidelity 2X PCR Master Mix manufacturer protocols (NEB) in a total reaction volume of 50 μl (1.25 μM each of i5 and i7 sequencing primers) at 72°C for 5 minutes, 98°C for 30 seconds, (98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute) x10 cycles. PCR samples were re-purified following the Zymo manufacturer protocol and eluted into 21 μl of elution buffer. Size selection was performed via agarose gel (150-1000 bp) followed by Qiagen MinElute gel extraction according to the manufacturer's instructions, with two serial 10 μl elution steps. Sequencing (75-bp paired-end) was performed on an Illumina NextSeq500.