GSM9033083: MNase-seq, Dox, 20U digestion, rep2; Mus musculus; MNase-Seq
5 × 10^6 cells in 4 ml medium were fixed with 100 µl 37% formaldehyde for 15 minutes at room temperature. The fixation was quenched by adding 1 ml 2.5 M glycine, and cells were pelleted by centrifugation at 600 g for 5 minutes at 4°C. Nuclei were isolated in NE buffer (20 mM HEPES, pH 7.9; 10 mM KCl; 0.5 mM spermidine; 0.1% Triton X-100; 20% glycerol with 1× protease inhibitors) and divided into five reactions (~ 1 million cells per reaction). Nuclei were resuspended in 150 µl wash buffer (20 mM HEPES-NaOH, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1× Halt protease inhibitor), and 3 µl 100 mM CaCl₂ was added. MNase was added in varying amounts (1 µl, 2.5 µl, or 5 µl corresponding to 10 U, 25 U, and 100 U, respectively) and incubated at 37°C for 5 minutes on a thermomixer. The reaction was stopped with 150 µl 2× STOP buffer. The digested DNA were incubate with RNaseA for 3 hours at 4°C and proteinaseK +SDS overnight at 55°C. The DNA were recoveried by PCI extraction and ethanol precipitation. 1µg recovered DNA were subjected to library preparation as described in CUT&RUN protocol. In brief, DNA were end-repair, add-A with Taq+T4 DNA polymerase+T4 PNK cocktails with 12°C 15 min, 37°C 15 min and 72°C 20min. The end-repaired DNA were ligated with barcoded TruSeq adapters with Quick ligase for 15 min at 20°C. Ampure XP bead (38 µl) were used to remove unligated adapters and P5/P7 primer were used for library amplification with KAPA-HS HiFi polymerase. Gel extraction was applied to purify library with 150-650bp size range.