HPC7 cell line stably expressing triple-FLAG-tagged murine MYSM1
HPC7 cell line
anti-H3K27Ac (Abcam, ab4729, Rabbit polyclonal)
Sequenced DNA Library
Cells were crosslinked for 10 min at room temperature with 1% formaldehyde added in culture medium. Crosslink was stopped with ice-cold PBS containing 0.125M glycine for 5min. Nuclei were prepared by sequential incubation on ice for 5min in buffer A [10mM Tris-HCl (pH 8), 10mM EDTA, 0.25% Triton X-100], and for 30min in buffer B [10mM Tris-HCl (pH 8), 1mM EDTA, 200mM NaCl] (all buffers include proteases inhibitors). Nuclei were resuspended in sonication buffer [10mM Tris-HCl (pH 8), 1mM EDTA, 0.5% SDS, 0.5% Triton X-100, 0.05% NaDOC, 140mM NaCl] and sonicated with a Branson Digital Sonifier (Branson Ultrasonics) to an average size of 250bp (12 cycles of 30 seconds at 80%, with 30 seconds rest in cooled circulating water). Sonicated chromatin from the equivalent of 5x10^6 cells per ChIP was incubated overnight on a rotating platform at 4°C with 40μL of Dynabeads Protein G (Invitrogen, Life Technologies) conjugated to 3-5μg of antibodies: anti-Flag M2 (Sigma, F1804, for MYSM1-FLAG ChIP), anti-H3K27Ac (Abcam, ab4729) or anti-H2AK119Ub (Cell Signaling Technology, D27C4, Table S10). Immune complexes with anti-Flag M2 antibody was washed sequentially for 2min at room temperature with 1ml of the following buffers: Wash 1 [0.5% NP-40, 150mM KCl, 1mM EDTA, 10mM Tris-HCl pH8]; Wash 2 [0.5% Triton, 0.1% NaDOC, 100mM NaCl, 10mM Tris-HCl pH8]; Wash 3A [0.5% Triton, 0.1% NaDOC, 400mM NaCl, 10mM Tris-HCl pH8]; Wash 3B [0.5% Triton, 0.1% NaDOC, 500mM NaCl, 10mM Tris-HCl pH8]; and Wash 4 [0.5% NaDOC, 0.5% NP-40, 250mM LiCl, 1mM EDTA, 10mM Tris-HCl pH8]. Immune complexes with anti-H3K27Ac and anti-H2AK119Ub antibodies were washed sequentially for 2min at room temperature with 1ml of the following buffers: Wash B [1% Triton X-100, 0.1% SDS, 150mM NaCl, 2mM EDTA, 20mM Tris-HCl pH 8]; Wash C [1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA, 20mM Tris-HCl pH 8]; Wash D [1% NP-40, 250mM LiCl, 1mM EDTA, 10mM Tris-HCl pH 8]; and TEN buffer [50mM NaCl, 10mM Tris-HCl pH 8, 1mM EDTA]. After de-crosslinking by overnight incubation at 65°C, the ChIP and Input DNA was purified with Qiaquick PCR Cleanup kit following manufacturer's instructions (Qiagen). ChIP-seq libraries were prepared using the Illumina TruSeq kit and sequenced on an Illumina HiSeq 2500 sequencer using a 50bp paired-end configuration. Sonicated input DNA from the same cells were sequenced as negative control.