GSM7433777: CR119_DA822_H3K9me3; Homo sapiens; OTHER
The nuclei isolation from the embryonic brain tissue was performed according to Jönsson et al., 2021. In brief, the tissue was thawed and dissociated in ice-cold lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a 1 ml tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose cushion (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before being resuspended in 300 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C at a low flow rate using a 100 mm nozzle (reanalysis showed >99% purity). CUT&RUN: We followed the protocol detailed by the Henikoff lab (Skene et al., 2017). Briefly, 100,000 sorted nuclei were washed twice (20 mMHEPESpH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x Roche cOmplete protease inhibitors) and attached to 10 ConA-coated magnetic beads (Bangs Laboratories) that had been pre-activated in binding buffer (20mM HEPES pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). Bead-bound cells were resuspended in 50 μL buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/vdigitonin, 2mM EDTA) containing primary antibody (rabbit anti-H3K4me3 Active Motif 39159, RRID:AB_2615077; or goat anti-rabbitIgG, Abcam ab97047, RRID:AB_10681025) at 1:50 dilution and incubated at 4°C overnight with gentle shaking. Beads were washed thoroughly with digitonin buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete protease inhibitors, 0.02% digitonin). After the final wash, pA-MNase (a generous gift from Steve Henikoff) was added to the digitonin buffer and incubated with the cells at 4°C for 1 h. Bead-bound cells were washed twice, resuspended in 100μL digitonin buffer, and chilled to 0-2°C. Genome cleavage was stimulated by the addition of 2mM CaCl2at 0°C for 30min. The reaction was quenched by the addition of 100μL 2x stop buffer (0.35M NaCl, 20mM EDTA, 4mM EGTA, 0.02% digitonin, 50ng/μL glycogen, 50ng/μLRNaseA, 10 fg/μL yeast spike-in DNA (a generous gift from Steve Henikoff)) and vortexing. After 10min of incubation at 37°C to releasegenomic fragments, cells and beads were pelleted by centrifugation (16,000 g, 5min, 4°C) and fragments from the supernatant were purified.