ChIP-seq of Nematostella vectensis: Adult
Adult animals (~1.2 g wet weight) were harvested and washed with PBS twice. The animals were treated with 2 % formaldehyde in PBS for 12 min at room temperature, and the cross-linking reagent was quenched with 0.125 M glycine for 5 min at room temperature. After washing with PBS twice, the crosslinked samples were resuspended in 10 ml buffer1 (50 mM Hepes, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM DTT, and protease inhibitors (Sigma)) and lysed with 10 slow strokes of a tight-fitting pestle (type B) in a 15 ml Dounce homogenizer. The lysate was centrifuged at 500 g for 5 min at 4 C, and the resulting pellet was resuspended in 10 ml Buffer1 and homogenized as described above. The homogenization processes were repeated 1-2 more times. In the last homogenization, the lysate was centrifuged at 2000 g for 10 min at 4 C, and the pellet (nuclei) was resuspended in 4 ml SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA,1% SDS, and protease inhibitors). The chromatin was sheared to 200-500 bp fragments by sonicating the samples 12 times (1 on and 1 off for 1 min) at an amplitude of 50% with a Branson Digital Sonifier. The sonicated samples were centrifuged at 21,000 g for 10 min at 4 C and then diluted 10X with CHIP dilution buffer (17.7 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS, and protease inhibitors). After the lysate was cleared with Protein A and G magnetic beads (Cell Signaling), 50 l of the cleared sample was set aside as input DNA, and 5 ml of lysate was incubated with 10 g anti-Brn3 Rabbit polyclonal antibody, which was conjugated to 30 l of protein A+G magnetic beads. After incubation at 4 C overnight, the beads were washed 3 times with 1 ml of Low salt buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), 3 times with 1 ml of High salt buffer (20mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS), 3 times with 1 ml of LiCl wash buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium dexycholate, and 1 mM EDTA), and 3 times with 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). The chromatin was eluted in SDS Elution buffer (50 mM Tris-HCl, pH8.0, 1% SDS, and 1mM EDTA), followed by reverse cross-linking at 65 C overnight. After being treated with RNase A (1 mg/ml) at 37 C for 1 h and then with protease K (0.2 mg/ml) at 45 C for 1 h, the DNA fragments were purified with QIAquick Spin columns (QIAGEN) and the purified DNA samples were quantified by Qubit4 (ThermoFisher). 20 ng of the immunoprecipitated DNA or input DNA was used to generate a library with the NEBNext Ultra II DNA Library kit following the manufacturers protocol. Libraries were initially quantified by Qubit4 and the size profiles were determined by TapeStation (Agilent) and then quantified by qPCR (NEBNext Library Quant Kit) for high-throughput sequencing. Biological replicates of libraries of immunoprecipitated DNA and the input DNA were pooled in equimolar ratio and the pooled libraries were sequenced on a DNBseq Sequencing platform (BGI, China) for PE 100 bp.