GSM9378584: KASseq_Rep2; Mus musculus; OTHER
two frozen testes from each mouse were dissected in cold 1×PBS and minced into small pieces after removing the tunica albuginea. The tiny tissue chunks were then homogenized into single cell suspension with a A-type Dounce glass homogenizer. After centrifugation for 3 min at 4°C, discarded the supernatant and suspended the cell pellet in 200 µL of labeling medium (5 mM N3-kethoxal in prewarmed 1×PBS) for 10 min at 37°C with gentle shaking (300 rpm), then harvest the cell pellet by centrifugation at 1200 g for 3 min. The total genomic DNA (gDNA) was isolated and finally eluted in 50 µL of 25 mM K3BO3 (pH 7.0) for each sample. For biotinylation, 2~3 µg of gDNA from each sample were mixed with 1 mM of DBCO-PEG4-biotin in 21 mM of K3BO3 (pH 7.0) in final 100 µL of 1×PBS buffer. The mixture was incubated at 37 °C for 1.5 h with shaking at 500 rpm to facilitate the click cycloaddition reaction, followed by RNA removal using RNase A digestion. The final DNA was purified by DNA Clean & Concentrator kit (Zymo) following the manufacturer's protocol and eluted in 50 µL of 25 mM K3BO3 (pH 7.0). Next, 1 μg of biotinylated DNA into 100 µL of 25 mM K3BO3 (pH 7.0) was sonicated to 150–350 bp sizes using Bioruptor Pico Sonicator (30s on/30s off setting for 22 cycles at 4 ℃). 5 µL of the fragmented DNA was saved as an input control. Subsequently, 10 µL of Dynabeads MyOne streptavidin C1 were washed three times with 50 µL of 1× B&W buffer (5 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 1 M NaCl and 0.05% Tween-20), using a magnetic rack to separate the beads after wash. Next, resuspended prewashed beads in 95 µL of 2× B&W buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 M NaCl and 0.1% Tween-20) with the 95 µL of fragmented DNA and incubated at room temperature for 20 min. To remove kethoxal, resuspended the beads in 15 µL of nuclease-free water and heated at 95 °C for 10 min to elute enriched ssDNA. The eluted DNA can be stored at −20 °C for subsequent ssDNA library construction following the manufacturer's protocol (Accel-NGS ssDNA library kit). The barcoded PCR was carried out for 12-14 cycles for each sample, and the library was subsequently size-selected for DNA ranging between 200 bp ~ 500 bp prior to PE150 sequencing.