GSM5007781: KU cytoDNA rep2; Mus musculus; OTHER
Primary cultured microglia were lysed in a lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100 plus protease inhibitors for 5 min on ice. Cell nuclei were removed by low-speed centrifugation (1500 g, 10 minutes). Cytoplasmic extracts were treated with 1 mg/ml Proteinase K at 56 °C for 2 hours. The aqueous supernatant was incubated with 500 mg/ml DNase-free RNase A (QIAGEN) for 30 minutes at 37 °C and followed by phenol/chloroform extraction. The DNA-containing aqueous phase was precipitated and resuspended in TE buffer. DNA concentrations were adjusted according to the protein concentration of cytoplasmic fractions and analyzed on a 2% agarose gel infused with ethidium bromide. The DNA concentrations were determined by Qubit Assays on a Qubit 4 Fluorometer. CytoDNA sequencing was performed on two biological replicates. Library preparation for cytoplasmic DNA was performed according to the KAPA Hyper Prep Kit (KR0961 – v6.17, KAPA biosystems) protocol, omitting the library amplification step to avoid sequence enrichment bias. The cytoplasmic DNA library was then analyzed on the Advanced Analytical Technologies Fragment Analyzer Automated CE System using an Agilent HS NGS Fragment Kit (Part # DNF- 474-0500), and size selected using AMPure XP beads (Beckman Coulter) for fragment sizes between 200-700 base pairs.