GSM5227091: E14-H3K27ac+RNAPII-rep2; Mus musculus; OTHER
Mouse ESCs or TSCs were trypsinized and counted using a TC-20 cell counter (Biorad). 100,000 cells were washed and resuspended in wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail) and used for bulk multi-CUT&Tag. 10 µL of Concanavalin A coated magnetic beads (Polysciences) were activated as described30 and added per sample and incubated at RT for 15 min. Bead-bound cells were resuspended in 100 µL Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 0.05% Digitonin; 1× Protease inhibitor cocktail) containing 2 mM EDTA and XX µg of each differentially barcoded purified antibody-pA-Tn5 complex was added. The mixture was incubated overnight at 4 °C for antibodies to bind. Cells were washed thrice in Dig-med Buffer (20 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM Spermidine; 0.05% Digitonin; 1× Protease inhibitor cocktail) by placing the tube on the magnet stand until the solution clears and removal of all of the liquid by pipetting. The cells were then resuspended in 300 µL Dig-med Buffer containing 10 mM MgCl2 and incubated at 37 °C for 1 h to activate tagmentation. To stop tagmentation, 10 µL of 0.5 M EDTA, 3 µL of 10% SDS and 1 µL of 20 mg/mL Proteinase K was added to each tube, which were incubated at 55 °C for 1 hour. DNA was extracted by performing one phenol:chloroform extraction followed by ethanol precipitation. The DNA pellet was resuspended in 22 µL of 10 mM Tris pH 8. The libraries were then amplified by mixing 21 µL of DNA with 2µL each of (10 µM) barcoded i5 and i7 primers, using a different combination for each sample. 25 µL NEBNext HiFi 2× PCR Master mix (NEB) was added, and PCR was performed using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 17 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min and hold at 4 °C. 1.1× volume of Ampure XP beads (Beckman Coulter) was incubated with libraries for 10 min room temperature to clean up the PCR mix. Bead bound DNA was purified by washing twice with 80% ethanol and eluting in 20 µL 10 mM Tris pH 8.0.