GSM9862422: Mouse sperm, DNase I, rep1; Mus musculus; ATAC-seq
After sperm counting, nuclei were isolated from 100,000 untreated or treated mouse sperm by centrifugation and lysis in buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂) containing 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin. Lysis was terminated with lysis buffer containing 0.1% Tween-20, and nuclei were pelleted by centrifugation. Purified nuclei were resuspended in the transposase reaction mixture containing 0.05% digitonin and incubated at 37°C for 35 min with shaking (1,000 rpm). Following transposition, genomic DNA was purified using proteinase K digestion followed by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation Libraries were PCR-amplified using the 2× KAPA SYBR FAST ABI Prism Kit (Kapa Biosystems) and 2.5 µM indexed primers under the following conditions: 72°C for 5 min, 98°C for 30 s, followed by 13 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min. Libraries were sequenced on an Illumina NovaSeq X Plus platform using 150-bp paired-end sequencing.