title: GSM5009778: CutTag_BBE2C-QC-H3K27me3-002; Homo sapiens; OTHER
library_construction_protocol: KDM4B CUT&Tag DNAs of BE2C cell with and without QC6352 treatment were prepared by following the protocol as described previously (Kaya-Okur et al. 2019 and https://www.protocols.io/view/bench-top-cut-amp-tag-bcuhiwt6?step=1 ) with minor modifications. BE2C cells approximately 500,000 with and without QC6352 200nM treatment were washed with wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail). Nuclei were isolated with cold NE1 buffer (20 mM HEPES–KOH, pH 7.9; 10 mM KCl 0.1%; Triton X-100; 20% Glycerol, 0.5 mM Spermidine; 1x Protease Inhibitor) for 10 min on ice. Nuclei were collected by 600 x g centrifuge and resuspended in 1ml washing buffer containing with 10 µL of activated concanavalin A-coated beads (Bangs laboratories, BP531) at RT for 10 min.  Bead-bound nuclei were collected with placing tube on magnet stand and removing clear liquid. The nuclei bound with bead were resuspended in 50 µL Dig-150 buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA) and incubated with a 1:50 dilution of KDM4B antibody (KDM4B, Active Motive, 61221) overnight at 4 °C. The unbound primary antibody was removed by placing the tube on the magnet stand and withdrawing the liquid. The primary antibody bound nuclei bead was mixed with Dig-150 buffer 100uL containing guinea pig anti-Rabbit IgG antibody (Antibodies, ABIN101961) 1:100 dilution for 1 hour at RT. Beads bound nuclei were washed using the magnet stand 3× for 5 min in 1 mL Dig-150 buffer to remove unbound antibodies.  A 1:250 dilution of pA-Tn5 adapter complex was prepared in Dig-300 buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.05% Digitonin, 1× Protease inhibitor cocktail).  After removing the liquid on the magnet stand, 100 µL mixture of pA-Tn5 and Dig- 300 buffer was added to the nuclei bound beads with gentle vortex and incubated at RT for 1 h. After 3× 5 min in 1 mL Dig-300 buffer to remove unbound pA-Tn5 protein, nuclei were resuspended in 250 µL Tagmentation buffer (10 mM MgCl2 in Dig-300 buffer) and incubated at 37 °C for 1 h. EDTA 10 µL of 0.5 M, 3 µL of 10% SDS and 2.5 µL of 20 mg/mL Proteinase K were added to stop tagmentation and incubated at 55 °C for 1 hour. DNA was then precipitated by phenol/chloroform/isoamylalcohol followed by ethanol precipitation with glycogen and then dissolved in water. Sequencing libraries were prepared using NEBNext HiFi 2× PCR Master Mix (NEB, M0541L) according to the manufacturer's instructions. The PCR products were cleaned up with SPRIselect beads and quantified using Qubit dsDNA HS assay kit (Agilent Technologies). The libraries were sequenced on a HiSeq2500 with paired-end 50-bp reads (Illumina).