GSM8521635: GATA2_Rep1; Homo sapiens; ChIP-Seq
150 million Ark1 cells were incubated in serum free media with 1% paraformaldehyde for 10 minutes at room temperature, then quenched with 125 mM glycine. Cells were scraped from the plate, pelleted for 5 minutes at 700xG, then resuspended in 9 mL of Buffer A (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and 1 tablet Pierce protease inhibitor cocktail mini) and incubated on ice for 10 minutes. The cells were then pelleted for 5 min at 1400xG, the supernatant was aspirated, and the pellet resuspended in 3 mL Buffer A. After incubation on ice for 5 minutes, cells were pelleted for 5 minutes at 1400xG and the supernatant was aspirated. The pellet was resuspended in 900 µL Buffer A, after which 100 µL of Buffer B (10 mM EGTA, 800 mM NaCl, 1% SOC, and 2% SDS) was added. The lysate was then split between two 1.5 mL Eppendorf tubes, and each lysate sonicated for 5 cycles at 30% amplitude for 15 seconds on and 30 seconds off in an ice water bath, using a Branson SLPe Digital Sonifier and 1/8-inch probe tip. Sonicated lysates were then spun at 14,000xG for 10 minutes and the supernatant transferred to a new 1.5 mL tube and glycerol added to 5% final concentration. For immunoprecipitations, 25 µg of chromatin was added to each tube to which 1 µg of rabbit anti-GATA2 or rabbit IgG control was added, followed by RIPA buffer up to 1 mL. For input, 2.5 µg of chromatin was added and antibody omitted. The tubes were then rotated overnight at 4 C. The next day, 30 µL of protein G magnetic beads (NEB) were washed with RIPA buffer and then split equally across IgG and GATA2 tubes, which were then rotated for 4 hours at 4 C. Using a magnetic tube rack, the beads were then washed 3X with 1 mL of RIPA buffer, 2X with 1 mL of RIPA buffer + 300 mM NaCl, 2X with 1 mL of LiCl buffer (250 mM LiCl, 0.5% NP-40, 0.5% DOC), and 1X with 1 mL TE buffer (10 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaCl). Beads then received 200 µL of ChIP Elution Buffer and tubes were heated overnight at 65 C shaking at 800 rpm with 30 seconds on and 10 second off. Using a magnetic rack, the supernatant was then collected and 20 µg RNase A was added followed by 1 hour incubation at 37 C. The samples then received 20 µg Proteinase K and were incubated for 55 C for 2 hours, followed by clean up using Qiagen PCR cleanup kit and elution into 50 µL EB buffer. Purified immunoprecipitated and input DNA was submitted to the University of Wisconsin-Madison Biotechnology Center. DNA concentration and sizing were verified using the Qubit® dsDNA HS Assay Kit (Invitrogen) and Agilent High Sensitivity D1000 ScreenTape (Agilent Technologies, Inc.), respectively. Samples were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs) with minor modifications from the manufacturer's protocol. Quality and quantity of the finished libraries were assessed using Agilent D1000 ScreenTape and Qubit® dsDNA HS Assay Kit, respectively. Libraries were standardized to 2nM. Paired end, 150 bp sequencing was performed using the Illumina NovaSeq X Plus (Illumina).