GSM7732659: P1137_MV411_cFOS_ChIP_FLT3i_Rep1; Homo sapiens; ChIP-Seq
Cells were treated with 10 nM gilteritinib or doxycycline to induce RASiDAb in the presence or absence of 10 ng/ml IL-3 for 24 h prior after which 20 million cells were harvested and crosslinked in 1% formaldehyde solution (methanol-free from Pierce, Thermo Scientific). 400 mM of glycine (Merck) was added, and cells were washed twice with PBS (Merck) after which pellets were frozen at -80 °C. Crosslinked cells were resuspended at 1x107 cells/ml in Buffer A (10 mM HEPES, 10 mM EDTA, 0.5 mM MEGTA, 0.25 % Triton- 100, 1x complete mini protease inhibitor cocktail (PIC) (Merck) pH 8.0) and incubated at 4 °C for 10 minutes prior to centrifugation at 500 xG for 10 min. This step was repeated with Buffer B (10 mM HEPES, 200 mM NaCl, 1 mM EDTA, 0.5 mM MEGTA, 0.01 % Triton-X 100, 1x PIC, pH 8.0) and after centrifugation 2 x 106 cells were resuspended in 300 μl IP buffer I (25 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100, 0.25 % SDS, 1x PIC, pH 8.0) and sonicated using a Diagenode Bioruptor Pico sonicator for 4 cycles (30 sec on 30 sec off) before centrifugation for 10 min at 16,000 xG. The supernatant was then collected and 600 μl IP Buffer II (25 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100, 7.5 % Glycerol, 1x PIC, pH 8.0) was added prior to immunoprecipitation. For immunoprecipitations 15 μl of Dynabeads-Protein G were washed twice with 500 μl 50 mM citrate phosphate buffer pH 5 and resupended in 15 μl citrate phosphate buffer with 4 μg anti-RUNX1 antibody (ab23980, Abcam) or (Invitrogen, MA5-15055) and 0.5% acetyl-BSA before incubation at 4 °C for 2 h. After incubation, dynabeads were washed with 500 μl pH 5 citrate phosphate buffer and resuspended in 15 μl citrate phosphate buffer with 0.5 % BSA before 555 μl of sonicated chromatin was added and incubated at 4 °C for ~ 16 h. After the incubation the dynabeads are washed sequentially with 500 μl of: Wash buffer 1 (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X 100, 0.1% SDS, pH 8.0) once, Wash buffer 2 (20 mM Tris-HCl, 500 mM NaCl, 20 mM EDTA, 1 % Triton-X 100, 0.1 % SDS, pH 8) twice, LiCl buffer (10 mM Tris-HCl,250 mM LiCl, 1 mM EDTA, 0.5 % NP-40, 0.5 % Na-deoxycholate, pH 8.0) once, TE/NaCl wash buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0) twice. After these washes DNA was eluted from the dynabeads using 100 μl elution buffer (100 mM NaHCO3, 1% SDS). 200 mM NaCl and 500 μg/ml proteinase K were added to the eluant and the sample was reverse crosslinked at 65 °C for >4h. DNA was then purified by ampure (1.8x). Libraries for next generation sequencing were prepared using a Kapa Hyper prep kit (Roche) according to manufacturers instructions. 16 cycles of PCR amplification were used and 200-450 bp fragments were size selected by gel electrophoresis. Libraries were validated by qPCR and quantified using the High Sensitivity DNA kit (Agilent) and Kapa Library Quantification kit (Roche) prior to sequencing on a Nextseq 2000 75 using a NextSeq 500/550 High output kit.