GSM6470212: IRF4_L428_2; Homo sapiens; ChIP-Seq
ChIP was globally performed as previously described (Cauchy et al 2015) using double-crosslinking. Cells were resuspended at 3.3x106 cells/ml in PBS and first crosslinked with 8.3l/ml DSG (Sigma) for 45 mins at room temperature, subsequently washed 4x and crosslinked with 1% formaldehyde for 10 mins at room temperature, with both crosslinking methods entailing sustained tube rotation. Crosslinking was quenched in 0.2M glycine and cells were washed 2x.  Cells were lysed in Buffer A (10 mM Hepes, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X100), then in Buffer B (10 mM Hepes, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01 % Triton X100), at 1x107 cells/ml and 4°C with rotation for 10 mins for both stages. Nuclei were resuspended at 2x107 cells/ml in 4°C IP Buffer I (25 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X100, 0.25% SDS) and sonicated in 6x 300 µl per reaction using a Picoruptor sonicator (Diagenode) at 240W with 30 cycles of 30s on, 30s off at 4°C. Cell debris was pelleted via 10 min 16,000g centrifugation and diluted in IP Buffer II (8.33 mM Tris, 50 mM NaCl, 6.33 mM EDTA, 0.33 % Triton X100 0.0833 % SDS, 5 % glycerol final concentration). 5% of chromatin was saved as input control. Immunoprecipitation was carried out overnight using Maximum Recovery tubes (Axigen) with rotation at 4°C in 50 µL PBS+0.02% Tween 20 with 15 µL protein G dynabeads that were washed blocked with 0.5% BSA and conjugated with either sc-6059-X (IRF4) or sc-46-X (JunB) antibodies for 4 hours at 4°C with rotation. Beads were subsequently washed on ice by magnetic separation using 1x PBS+0.02% Tween 20, 2x Wash Buffer 1 (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 1 % Triton X100, 0.1 % SDS), 1x Wash Buffer 2 (20 mM Tris, 500 mM NaCl, 2 mM EDTA, 1 % Triton X100, 0.1 % SDS), 1x with LiCl Buffer (10 mM Tris, 250 mM LiCl, 1 mM EDTA, 0.5 % NP40, 0.5 % Na-deoxycholate), 2x with TE/NaCl Buffer (10mM Tris, 50 mM NaCl, 1 mM EDTA). Beads were eluted using 2x 50 µl Elution Buffer (100 mM NaHCO3, 1% SDS) with shaking for 15 min at RT and eluates were pooled. Chromatin was reverse-crosslinked overnight at 65 °C in Elution Buffer + 200 mM NaCl, followed by 100 µg/ml Rnase A and 0.25 mg/ml proteinase K digestion for 1h at 37 °C and 55 °C, respectively. DNA was purified using via phenol-chloroform extraction. ChIP efficiency was checked using IL3 -40, CSF1R FIRE, and gene desert control oligonucleotides (Bevington et al., 2016). Library preparation was performed using the KAPA hyperprep kit (Roche) following manufacturer's guidelines. Libraries were sequenced in single-end mode at 50 million reads per library using an Illumina HiSeq 2000 system according to manufacturer's instructions.