GSM6255813: WT K562, IgG, EpiCypher, rep2; Homo sapiens; OTHER
Chromatin profiling for WT and ACK KO K562 cells was performed using CUTANA™ ChIC/CUT&RUN Kit (EpiCypher) according to the manufacturer's protocol . Wash Buffer, Cell Permeabilization Buffer and Antibody Buffer were supplemented with 1% Triton X-100 and 0.05% SDS. Cells were washed twice with wash buffer, and resuspended in 410ul wash buffer. 100 µL of this solution was aliquoted into four reaction tubes i.e. 5 x 107 cells per CUT&RUN reaction. 100 µL of pre-activated ConA bead slurry was added to each reaction tube and the mixture incubated for 10 min at room temperature to allow cell-bead binding. Cell-bead solution was then buffer exchanged into Antibody Buffer and target-specific antibodies added to individual reaction tubes in the following amounts: 0.5 µg of anti-H3K4me3, 1 µg of anti-H3K27ac , 0.6 µg of anti-Stat5 and 0.5 µg of IgG as a negative control.  Antibody binding was performed at 4oC overnight with gentle mixing on a nutator. Binding of pAG-MNase, targeted chromatin digestion and DNA release were completed according to the manufacturer's protocol. To reverse cross-links, CUT&RUN released DNA was supplemented with 0.8 µL of 10% SDS (Merck) and 1 µL of 20 µg/ul Proteinase K (Ambion), and incubated overnight at 55oC using a thermocycler. DNA purification was performed using DNA Cleanup Columns. CUTnRUN libraries were prepared using the KAPA hyperprep kit (Roche) and multiplexed using 1.5uM custom TruSeq UDI-UMI adapters. Libraries were amplified using 14 cycles on the thermocycler.  Libraries were quantified on a fragment analyzer 5300 system (Agilent) and pooled equimolarly to a final 1.9 nM concentration and sequences using a 51bp pared end setting on a Illumina NovaSeq6000.