title: GSM6281806: H3K27ac_ESC_500_rep1; Mus musculus; OTHER
library_construction_protocol: CUT&RUN libraries were generated based on the original protocol (Meers et al, 2019) with some modifications to optimize for low-input cell numbers. For each library, 10μl BioMag Plus Concanavalin A beads (Polysciences) were washed twice in 1ml Binding Buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2) and resuspended in 5μl Binding Buffer. Then embryos were added in 45μl Wash Buffer (20mM HEPES-NaOH pH7.5, 150mM NaCl, 0.5mM Spermidine, 1X Roche Protease Inhibitor Cocktail), mixed with the 5μl Binding Buffer containing beads and rotated for 10min at room temperature. After removing the liquid by putting the tube on the magnet stand, the beads were resuspended in 80μl antibody Incubation Buffer (1X Wash Buffer with 0.02% Digitonin and 2mM EDTA) with 1:100 diluted H3K27ac antibody (Millipore, 07-360) and rotated at 4oC overnight. Then the beads were washed twice with 100μl Wash Buffer with 0.02% Digitonin and pulled off the liquid. The beads were resuspended in 100μl Wash Buffer with 0.02% Digitonin and 500ng/ml pA-MNase, then rotated for 3h at 4oC. After washing the beads twice with 200μl Wash Buffer with 0.02% Digitonin, the beads were resuspended in 200μl ice-cold Wash Buffer with 0.02% Digitonin and 4μl 100mM CaCl2 and incubated at 0oC for 30min. The reaction was stopped by adding 23μl 10X Stop Buffer (1,700mM NaCl, 100mM EDTA, 20mM EGTA, 0.02% Digitonin, 250µg/ml RNase A, 250µg/ml Glycogen). Then the tube was incubated at 37oC for 10min to release the cut fragments into supernatant. The tube was put on a magnet stand and the supernatant was transferred to a new 1.5ml LoBind tube (Eppendorf) while the beads were discarded. 2.5μl 10% SDS and 2.5μl Proteinase K (20mg/ml) were added to the supernatant and incubated at 55oC for 1h. Then 25ng carrier RNA (Qiagen, 1068337) was added to the supernatant. The supernatant was mixed with 200μl Phenol:Chloroform:Isoamyl Alcohol (Invitrogen, 15593031) by vortexing then transferred to a phase-lock tube (QuantaBio, 2302830) and centrifuged at 16,000g for 5min at room temperature. The supernatant was transferred to a new 1.5ml LoBind tube, added 26μl 3M Sodium Acetate, 1μl Glycogen (20mg/ml) and 750μl cold 100% Ethanol, mixed by vortexing and stored at -20oC overnight. Then the tube was centrifuged at 16,000g for 20min at 4oC and the supernatant was discarded. The precipitate DNA pellet was washed with 1ml cold 80% Ethanol then centrifuged at 16,000g for 5min at 4oC and the supernatant was discarded. Finally the precipitate DNA pellet was dissolved in 25μl ultrapure H2O. The library was constructed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) following the manufacturer protocol.