title: GSM3208743: MUT_2_p53-ChIP; Danio rerio; ChIP-Seq
library_construction_protocol: Approximately 1,000 of each WT and MUT supt16h embryos per treatment were collected and deyolked using the Ginzburg Fish Ringer solution. Embryos were resuspended in 1 mL PBS with 1 mM PMSF, 1X cOmplete EDTA Free Protease Inhibitor Cocktail Tablet (Roche), 1X phosSTOP Phosphatase Inhibitor Tablet (Roche) and pushed through a 21G needle. Embryos were immediately crosslinked with 4 mM DSG (ProteoChem) for 30 min, followed by 1% PFA for 8 min, and stopped with 0.125M Glycine. Fixed embryos were resuspended in 1 mL Lysis Buffer 1 (0.05M HEPES, 140 mM NaCl, 1 mM EDTA, 1% Glycerol, 0.5% NP40, 0.25% Triton-X) and subjected to the TissueRuptorII (Qiagen) for 20 sec on low. Tissue was rocked for 10 min at 4ºC, pelleted, and resuspended in 1 mL Lysis Buffer 2 (10 mM Tris,-pH 8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA). They were rocked again for 10 min at room temperature (RT), pelleted and resuspended in 1.5 mL Lysis Buffer 3 (10mM Tris-pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.05% SDS). Tissue was then sonicated on the Diagenode Bioruptor 300 (30 sec ON, 30 sec OFF, HIGH, 34 cycles). Following the addition of 0.8% Triton-X to the sonicated chromatin, cells were spun down. 20 ul of lysate was saved as input control and the remainder was incubated with Dyna Beads bound with 10 ug p53 antibody (GeneTex) overnight at 4ºC, rotating. Beads were collected on a magnet and washed 1x Wash Buffer 1 (20 mM Tris-pH 8, 150 mM NaCl, 2 mM EDTA. 0.1% SDS, 1% Triton-X), 1x Wash Buffer 2 (20mM Tris-pH 8, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X), 1x Wash Buffer 3 (10mM Tris-pH 8, 250 mM LiCl, 1 mM EDTA, 1% sodium deoxycholate, 1% NP40), 3x TET Buffer (1x TE, 0.2% Tween-20), and 1x TE-NaCl (1x TE, 50 mM NaCl). Beads were then resuspended in 500 ul Elution Buffer (50mM NaHCO3, 1% SDS) and incubated overnight with 0.2M NaCl at 65ºC to reverse crosslinking. Lysate was then incubated for 2 hrs at 37ºC with 5 ul RNase A (10 mg/ml) and 2 hrs at 55ºC with 5 ul Proteinase K (20 mg/ml). DNA was purified using Phenol/Chroloform/Isoamyl and resuspended in 50 ul TE (10 mM Tris-pH 8, 1 mM EDTA). TruSeq ChIP Library Preparation Kit (Illumina) was used to construct libraries which were subsequently run at SR75 on the HiSeq4000.