ChIP-Seq of human: adult male bone_marrow tumor: Sample H_KA-375182-h3k4_mono
"ChIP-seq was performed with the MAGnify ChIP kit (Thermo Fisher) using a total of 1.5 million cryopreserved primary AML cells per sample, which were distributed across immunoprecipitation reactions for histone H3K4 mono-methylation (Abcam, ab8895), H3K4 tri-methylation (Abcam, ab8580), and H3K27 tri-methylation (Active Motif, 39155). Antibodies were prepared by incubating with either protein A or protein G beads (5 ug per antibody) at 4oC for >1 hour. Cells were pelleted, resuspended in 500 uL PBS + protease inhibitor, and then crosslinked with 16% formaldehyde (1% final concentration) for 10 minutes at room temperature. Crosslinking reaction was stopped with 0.125 M glycine for 5 minutes at room temperature, washed twice with 500 uL cold PBS + protease inhibitor, and then resuspended in 50 uL ChIP lysis buffer + protease inhibitor for each ChIP antibody used. Lysates were then sheared on a Covaris S220 (duty cycle 5%, PIP 70%, cycles/burst 200, 60 second cycle time for 12 cycles), followed by centrifugation at >12,000g for 5 minutes to remove cellular debris. A portion (5 uL) of the supernatant was removed for shear analysis, and the remaining cleared lysate was split equally for each immunoprecipitation, diluted in dilution buffer + protease inhibitor, and added to antibody-bead conjugates in a total volume of 400 uL for bead-based immunoprecipitation with the above antibodies overnight at 4oC. Immunoprecipitated material was washed twice in IP buffers (buffer 1 then 2), treated with RC buffer and proteinase K to reverse crosslinking, and immunoprecipitated DNA was bead-purified. Library molecules were prepared using the NEBNext Library Prep Kit for Illumina, which involved enzymatic end repair, A-tailing, adapter ligation, USER enzyme treatment, and amplification with sample-specific index sequences, as described in the protocol. "