GSM6235356: H4k20me1, 8hrs, Rep1; Hydra vulgaris; ChIP-Seq
Two thousand Hydra polyps per time point were decapitated and the regenerating tips were collected and cross-linked with 1% methanol free formaldehyde. The fixation reaction was quenched with 150 mM Glycine. The fixed regenerating tips were resuspended in Swelling Buffer (25 mM Tris-HCl pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, 1X Protease inhibitor cocktail/PIC) and homogenized using a Dounce homogenizer (35 strokes) to lyse the cells and release the nuclei. The nuclei were pelleted, resuspended in sonication buffer (50 mM Tris-HCl pH 7.9, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 1% SDS, 0.1% Sodium deoxycholate, 0.5 mM PMSF, 1X PIC), and incubated for 30 min on ice. They were then sonicated to obtain an average chromatin size of 300 bp. Chromatin was pre-cleared using 50 μl of a 50% protein A sepharose (GE healthcare) slurry for 1 h at 4°C with gentle inverting. Immunoprecipitations were carried out in ChIP buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X 100, 0.01% SDS, 1X PIC) with Anti-H4K20me1. An appropriate IgG control was also used with inverting at 4°C for 14–16 h. The samples were then incubated with 50 μl of a 50% Protein A sepharose slurry (saturated with 0.5% BSA and 10 mg/ml yeast tRNA) for 3 h at 4°C with gentle inverting. ChIP samples were reverse-crosslinked, and the DNA was purified using Phenol:Chloroform:Isomamyl alcohol based precipitation method. Input chromatin was obtained after pre-clearing, by de-crosslinking and purifying input DNA Phenol:Chloroform:Isoamyl alcohol based precipitation method. Purified DNA was subjected to library preparation for sequencing A total of 1 ng of purified ChIPped DNA for each sample was used for library preparation using Ultra II DNA kit (NEB) as per the manufacturer's instructions. All library samples were amplified for 9-10 cycles depending on cycle number estimation by qPCR. Amplified libraries were subjected to double-sided bead purification using one round of 0.5X volume of Hi-prep PCR purification kit (Magbio Genomics, USA) to remove the primer dimers and one round of 1.8X volume to remove the fragments larger than 1000 bp. Library concentration was determined using Qubit (Thermo scientific, USA), and average fragment size was estimated using DNA HS assay on Bioanalyzer 2100 (Agilent Technologies, USA) before pooling libraries at equimolar ratio.