GSM8764133: G1_ESC_DMSO_Micro-C_rep6; Mus musculus; OTHER
The pellet was resuspended in 200 µL of MB1 buffer (50 mM NaCl, 10 mM Tris-HCl pH7.5, 5 mM MgCl2, 1 mM CaCl2, 0.2% v/v NP-40 alternative (MilliporeSigma, 492016), 1 X cOmplete EDTA-free protease inhibitor cocktail in ddH2O) and incubated on ice for 20 min. The samples were centrifuged at 1,750 × g for 5 min at 4˚C, and the pellets were washed with 200 µL of MB1 buffer. The nuclei pellets were then resuspended in 200 µL of MB1 buffer, and 28 U of MNase (Worthinigton Biochemical Corporation, LS004798) was added. After brief vortexing, the samples were incubated at 37˚C for 20 min on a shaker at 1,000 rpm. The amount of MNase to use was determined based on a separate titration experiment. The samples were transferred onto ice and 1.6 µL of 500 mM EGTA was added to stop the digestion. The reaction was further incubated at 65˚C for 10 min for complete inactivation of MNase. The samples were centrifuged at 170 × g for 5 min at 4˚C, and the pellets were washed with 1 mL MB2 (50 mM NaCl, 10 mM Tris-HCl pH 7.6, 10 mM MgCl2 in ddH2O). The pellets were resuspended in 50 µL of 1X Nuclease Digestion Buffer from Dovetail Micro-C kit. To lyse the nuclei, 3 µL of 20% v/v SDS was added, and the samples were incubated at 22˚C for 15 min on a shaker at 1,250 rpm. The rest of the steps in Micro-C library preparation, including lysate quality control, proximity ligation, library preparation, ligation capture, and library amplification, were performed following Dovetail Micro-C kit user guide v1.2.