GSM6318297: E14, WT, primed, replicate 2, CUT-RUN, IgG; Mus musculus; OTHER
CUT-RUN for MLL3/4 and H3K4me1 in E14 WT and mutant ES cells was done using CUTANA ChIC/CUT&RUN kit (Epicypher #14-1048) following manufacturer's protocol. Briefly, 5 × 105 cells were captured with Concanavalin A, permeabilized using 0.01% digitonin and incubated with 0.5 μg antibody (anti-MLL3/4 serum [kind gift from Dr. Joanna Wysocka] / anti-H3K4me1 [# 13-0040; Epicypher]/ IgG [# 13-0042; Epicypher]) in 50 μL antibody buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x protease inhibitor cocktails [EDTA-free tablet; Roche], 2 mM EDTA, 0.01% digitonin) for overnight. After removing unbound antibody, pAG-MNase (20X) in 50 μL cell permeabilization buffer was added to the cells and incubated for 10 min at RT. Then CaCl2 (2 mM) was added to activate MNase and incubated at 4 °C for 2 hr. The reaction was stopped using 33 μL of 2X STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 50 µg/mL glycogen) and E. coli spike-in DNA (0.5ng) added as a control. The DNA from the released chromatin in the supernatant was purified and then quantified using Qubit dsDNA HS assay kit (Agilent Technologies). CUT-RUN libraries were constructed using NEBNext Ultra II DNA library preparation kit. Briefly, end repair and dA-tailing were conducted on 6 ng of CUT&RUN eluted DNA for 30 min at 20°C followed by 30 min at 65°C. After adaptor ligation for 30 min at 20°C, the DNA fragments were purified by 1X vol of AMPure XP beads (Beckman Coulter) followed by 10-12 cycles of PCR amplification with Next Ultra II Q5 master mix. The PCR products were purified with 1X vol of AMPure XP beads.