GSM6042847: wt_p7_pu1; Mus musculus; ChIP-Seq
20 million HPC5 cells were fixed with 1% formaldehyde (Sigma-Aldrich; St. Louis, MO) for 10 minutes at room temperature with gentle stirring. Fixing was quenched with 2M glycine (Sigma-Aldrich; St. Louis, MO) and further stirring for five minutes at room temperature. Cells were collected by centrifugation and snap frozen for storage at -80°C. Cells were then resuspended in buffer L1 (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) supplemented with one Protease Inhibitor tablet (Roche; Basel, Switzerland). During lysis, samples were kept on ice for 10 minutes with gentle agitation every two minutes. Samples were centrifuged at 1693 rcf for 10 minutes at 4°C in a swing bucket rotor. Supernatant was removed and cells were resuspended in buffer L2 (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA, 10mM Tris pH8.0) supplemented with one Protease Inhibitor tablet (Roche; Basel Switzerland). Samples were rocked at room temperature for 10 minutes. Nuclei were isolated by centrifugation at 1693 rcf for 10 minutes at 4°C. Supernatant was carefully removed and 1X RIPA buffer was added to samples. Nuclei were sonicated using a cell disruptor (SFX250) (Branson Ultrasonics Corporation; Brookefield, CT). Lysate was clarified with centrifugation at 18879 rcf for 15 minutes at 4°C. The supernatant was carefully transferred to a 15mL conical vial. Samples were incubated with protein A/G sepharose beads (Pierce; Waltham, MA) overnight to preclear supernatant. 20µg of anti-PU.1 (sc-390405, Santa Cruz; Dallas, TX), and anti-NFIX (clone: 7B5.3) antibodies were also pre-bound to protein A/G agarose beads (Pierce; Waltham, MA) for overnight incubation. Pre-cleared chromatin was centrifuged at 1000 rcf for three minutes at 4°C. Pre-cleared chromatin was added to respective antibody-bead complex in Eppendorf tubes while some pre-cleared chromatin was reserved for input. Samples were rotated at 4°C for four hours. After incubation, samples were centrifuged at 5418 rcf for five minutes at 4°C. Supernatant was aspirated and beads were washed with various buffers: once with IP Wash Buffer I (20mM Tris pH8.0, 50mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), twice with IP High Salt Wash Buffer (20mM Tris pH8.0, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), once with IP Wash Buffer II (10mM Tris pH8.0, 250mM LiCl, 1mM EDTA, 1% NP-40, 1% deoxycholic acid), and twice with TE buffer. DNA:protein complexes from beads were eluted twice with freshly made elution buffer (1% SDS, 100mM sodium bicarbonate). To reverse crosslinking, 5M NaCl (final concentration 370mM), 10mg/mL RNase (final concentration 0.45mg/mL), and 20mg/mL proteinase-K (final concentration 0.26mg/mL) were added to eluates and input samples. Incubate samples at 37°C for 30 minutes, 45°C for 30 minutes, and 65°C overnight. DNA was eluted using the Qiagen MinElute (Hilden, Germany) kit following manufacturer's protocol except DNA was eluted in 28uL with provided EB buffer. DNA was quantified using the Quant-iT PicoGreen assay (Life Technologies; Carlsbad, CA) Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific; Waltham, MA) or SpectraMax Quant AccuBlue Pico dsDNA assay kit (Molecular Devices; San Jose, CA). Libraries were prepared with KAPA HyperPrep Library Preparation Kits (KK8504) (Roche; Basel, Switzerland). Libraries were analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent Technologies; Santa Clara, CA), 5300 Fragment Analyzer System HS Large Fragment Kit (Agilent Technologies; Santa Clara, CA), 4200 TapeStation D1000 ScreenTape assay (Agilent Technologies; Santa Clara, CA) or Caliper LabChip GX DNA High Sensitivity Reagent Kit (PerkinElmer; Waltham, MA). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (Life Technologies; Carlsbad, CA) or low pass sequencing with a MiSeq nano kit (Illumina; San Diego, CA). Single read 50 cycle sequencing was performed on a NovaSeq 6000 (Illumina; San Diego, CA) or NextSeq 550 (Illumina; San Diego, CA).