GSM9023459: ChIP_A375_NT_2_HMGB2; Homo sapiens; ChIP-Seq
For profiling of HMGB2 binding in A375 and SKMEL5 cells, freshly harvested cells (~20 million cells/replicate/condition) were cross-linked first with 1.5 mM EGS (Thermo Fisher 21565) for 20 min at room temperature, and subsequently with 1% formaldehyde (Thermo Fisher 28906) for 40 min at 4°C. The reaction was quenched by the addition of glycine to the final concentration of 0.125 M. Fixed cells were washed twice with PBS and resuspended in SDS buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% SDS, 1x protease inhibitor cocktail, Roche). The resulting nuclei were spun down, resuspended in the immunoprecipitation buffer (100 mM NaCl, 100 mM TrisHCl pH 8.0, 5 mM EDTA, 5% Triton X-100) at 1 mL per 0.5 million cells mixed in 2:1 ratio, with the addition of 1x protease inhibitor cocktail (Millipore Sigma 11836170001). Nuclei were processed on a Covaris E220 Focused-ultrasonicator to achieve an average fragment length of 200-300 bp with the following parameters: PIP=140, Duty Factor=5, CBP/Burst per sec=200, Time = 20 min (for A375 cells) or 45 min (for SKMEL5 cells). Chromatin concentrations were estimated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher 23227) according to the manufacturer's instructions. The immunoprecipitation reactions were set up in 500 µL of the immunoprecipitation buffer in Protein LoBind tubes (Eppendorf 22431081) and pre-cleared with 50 µL of Protein G Dynabeads (Thermo Fisher 10004D) for two hours at 4°C. After pre-clearing, the samples were transferred into new Protein LoBind tubes and incubated overnight at 4°C with 5 µg of HMGB2 (abcam ab67282), V5 (abcam ab9116, used for SKMEL5 ChIP only) and H3K4me3 (Epicypher 13-0041) antibodies. For normalization purposes, 5 µL of Drosophila spike-in chromatin (Active Motif 53083) and 2 µL of spike-in antibody (Active Motif 61686) were added to each reaction. The next day, 50 µL of BSA-blocked Protein G Dynabeads were added to each reaction and incubated for 2 hours at 4°C. The beads were then washed two times with low-salt washing buffer (150mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), two times with high-salt washing buffer (500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), two times with LiCL wash buffer (250 mM LiCl, 10 mM TrisHCl pH8.0, 1 mM EDTA, 1% Na-deoxycholate, 1% IGEPAL CA-630) and one time with TE buffer (10 mM TrisHCl pH 8.0, 1 mM EDTA). The samples were then reverse-crosslinked overnight in the elution buffer (1% SDS, 0.1 M NaHCO3) and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research D5205) following the manufacturer's instructions. After quantification of the recovered DNA fragments, libraries were prepared using the ThruPLEX®DNA-Seq kit (Takara R400676) following the manufacturer's instructions, purified with SPRIselect magnetic beads (Beckman Coulter B23318), and quantified using a Qubit Flex fluorometer (Thermo Fisher) and profiled by TapeStation (Agilent). The libraries were sent to MSKCC Integrated Genomics Operation core facility for sequencing on an Illumina NovaSeq 6000 (~30-40 million 100 bp paired-end reads per library).