GSM6066072: dox_wt_ChIP; Mus musculus; ChIP-Seq
EBF1 ChIP was performed as previously described (Boller et al. 2016) with minor changes. For spike-in normalization, murine pro-B cells were mixed with the EBF1-expressing human B cell lymphoma cell line BJAB in a 10/1 ratio to simultaneously immunoprecipitate murine and human EBF1 with the same antibody. Cells were harvested, washed with PBS and resuspended in PBS with 2% FCS (4x10^6 cells per 1 ml). formaldehyde (FA) mix (50 mM HEPES-KOH pH 8.0, 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 11% (v/v) formaldehyde) was added to a final concentration of 1% FA. Tubes were incubated on a rotator for 10 min at RT. Quenching was performed by adding 2 M glycine to a final concentration of 0.2 M. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 1% (w/v) SDS, 10 mM EDTA; PIM) (4x10^6 cells per 100 μl). Chromatin was sheared with a Bioruptor (25 cycles, output level "High", 30 sec ON, 30 sec OFF, 4°C) and fragment size and DNA concentration were checked. 20 μg DNA was used per ChIP reaction. Chromatin was mixed with 4 µg a-EBF1 (1C) rabbit antibody (Roessler et al., 2007) or rabbit IgG and incubated on rotator over night at 4°C. Chromatin was incubated with 30 μl Protein G Dynabeads suspension (ThermoFisherScientific) per sample on a rotator for four hours at 4°C. Beads were washed four times with wash buffer A (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2 mM EDTA) and once with final wash buffer A (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2 mM EDTA) for 5 minutes on a rotator at 4°C. Chromatin was eluted with 100 μl elution buffer (10 mM Tris-HCl, pH 8.0; 0.5% SDS; 300 mM NaCl; 5 mM EDTA pH 8.0) by shaking vigorously at 65 °C for 30 min. Samples were reverse crosslinked, treated with 10 µg RNase A and 10 µg Proteinase K, and purified using the QIAquick PCR Purification Kit. Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) according to the manufacturer's protocol.