GSM1486388: P60 Cerebellum DNase-seq Rep 2; Mus musculus; DNase-Hypersensitivity
15-30 million nuclei were digested with a range of recombinant DNase I enzyme (Roche) concentrations (between 1.2 and 12U) for 15 minutes at 37°C in 120uL 1X DNase buffer. Digestions were checked by pulse field gel electrophoresis and material was pooled from 3 different DNase concentrations (extent of digestion matched between samples) in equimolar amounts following blunt-ending reactions. This was followed by ligation to adapters, MmeI digestion, streptavidin bead-based enrichment, 14 cycles of PCR amplification, and separation from linker-only bands on a gel. C57BL/6Ncrl male and female mice at P7, P14, or P60 were deeply anesthetized with isofluorane and decapitated for brain harvesting. The cerebellar cortex was dissected, flash frozen, and stored at -80°C. Each cerebellum was dounced in 5mL 2M sucrose, 1mM MgCl2 and poured through a 100μM filter. Filtered tissue was ultracentrifuged in 15 mL of 2M sucrose, 1mM MgCl2 at 65,000 x g for 80 min. Supernatant was removed and the tight nuclear pellet was resuspended by pipetting in 1 mL buffer RSB from the DNase-seq protocol. Nuclei were quantified by hemocytometer, yielding approximately 5-10 million per adult cerebellum (~30 mg tissue).
GEO Accession
GSM1486388