GSM3581646: P33T-PP_H3K27ac_Lane3_TAGCTT; Homo sapiens; ChIP-Seq
PDX1P33T/+ PP cells (2 × 106 cells) were cross-linked in 1% formaldehyde in culture medium for 10 min at room temperature. The cross-linking reaction was stopped by the addition of glycine to a final concentration of 125 mM. For chromatin fragmentation, cells were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS) and sonicated in a Covaris S220 sonicator with a duty cycle of 2%, a peak incident power of 105 W and 200 cycles per burst for 20 min. The fragmented chromatin was diluted 1:5 in IP-Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-Desoxycholate, 140 mM NaCl, H2O, Protease Inhibitors) and directly used for immunoprecipitation. For PDX1 ChIP, 60% of the chromatin (equivalent of 1.2 × 106 cells) was processed in two parallel ChIPs using magnetic beads, preloaded with 3 µl goat anti PDX1 antibody (kindly provided by C. Wright), for each ChIP. For H3K27ac ChIP, 40% of the chromatin (equivalent 0.4 × 106 cells) was processed in three parallel IPs using magnetic beads, preloaded with 3 µg anti H3K27ac antibody (Diagenode, pAb-174-050). For all ChIPs, antibody incubation was performed at 4 °C for 5 h. Beads were then washed 5 times using (1.) IP-Buffer, (2.) Washing-Buffer 1 (500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% NP-40), (3.) Washing-Buffer 2 (250 mM LiCl, 50 mM Tris-HCl (pH 8.0), 0.5% Na-Deoxycholate, 1% NP-40) and (4. & 5.) Washing-Buffer 3 (10 mM Tris-HCl (pH 8.0), 10 mM EDTA). Subsequently, protein-DNA complexes were eluted from the beads in Elution-Buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS) at 65 °C for 20 min. Cross-links were reversed at 65 °C overnight and DNA was purified by ethanol precipitation. Libraries were constructed using the MicroPlex kit (Diagenode, C05010010). Library construction, purification and size selection using AMPureXP (Beckman Coulter) were performed according to the MicroPlex kit instruction manual (version 4 / 03.09.14)