title: GSM5915360: OCI-AML3 NPM1c degron2 cell line treated with dTag-13-24hr for Cut&Run of H3K27me3; Homo sapiens; OTHER
library_construction_protocol: Cut & Run and RNase Cut & Run were performed with nuclear extraction. Basically, 0.5 to 2 X106 cells were harvested and extracted for the nucleus using nuclear extraction buffer (20mM HEPES pH 7.9, 0.5mM Spermidine, 10mM KCl, 0.1% Triton-X, 20% Glycerol with Protease inhibitor) on ice for 5-10min. After washing with Wash Buffer 2 (20mM HEPES pH 7.5, 0.5mM Spermidine, 0.1% BSA, 150mM NaCl with Protease inhibitor), nuclei were incubated together with activated Concanavalin A Beads (Bang Labs) for 10 minutes at room temperature. Nuclei were then washed incubated with antibodies at 4℃ with 35 rpm rotation for 2 hours (H3K4me3, H3K27me3, Menin, XPO1), 4 hours (NPM1c, NPM1-WT), or overnight (RNA Pol II). pA/G-MNase (Epicypher) or pA-MNase (Gift from Dr. Steve Henikoff ) were then incubated with the nuclei-bead mixture in Wash Buffer 2 at 4℃ with 35 rpm rotation for 1hour. After washing with Wash buffer 2 for two times, CaCl2 was added to nuclei to activate the MNase to release chromatin. Stop buffer (340mM NaCl, 20mM EDTA pH 8.0, 4mM EGTA, 50μg/mL RNase A, 50μg/mL Glycogen) was added to stop the MNase and digestion of chromatin was performed with 5uL proteinase K at 55℃ overnight. DNA was extracted using Phenol:Chloroform:Isoamyl Alcohol (Sigma) extraction and MaXtract High Density tube (Qiagen). The aqueous phase was taken, and DNA was precipitated with 1/10 volume of 3M Sodium Acetate, pH 5.2, and 2.5 volume of 100% Ethanol at -20°C overnight. DNA was precipitated by 30min centrifugation at 4℃ and washed with 70% cold ethanol. Libraries were made using Swift Bioscience Acce-NGS 2G plus kit according to the manufacturer's manual. The library was sequenced 150bp paired-end on HiSeq2000 by Fulgent Genomics. Accel-NGS® 1S Plus  DNA Library Swiftbio Science (IDT XGEN DNA ssDNA low input since 2022 )