Illumina HiSeq 4000 paired end sequencing; ChIP-seq for NF-YA and Pbx4 on whole zebrafish embryos at 3.5hpf
ChIP-seq for NF-YA and Pbx4 on whole zebrafish embryos at 3.5hpf
Groups of ~5,000 embryos (for Pbx4) and ~10,000 embryos (for NF-YA) were collected at 3.5hpf and dechorionated in 1X pronase. The embryos were then dissociated by pipette, fixed in 2% formaldehyde in PBS for 10 minutes at room temperature, quenched with 125mM glycine, and flash-frozen in liquid nitrogen. Purification of the DNA was accomplished using the MicroChIP Dia Pure Column kit (Diagenode) according to the manufacturer's guidelines with an 11µL elution. To quantify the concentration of DNA, 1µL of each sample was passed through the dsDNA HS Assay (ThermoFisher Scientific) according to the manufacturer's guidelines and quantified on a Qubit device. Processing of cell pellets followed the protocol previously described (Amin et al., 2015). Nuclei were isolated in L1 Buffer (50mM Tris-HCl pH 8.0, 2mM EDTA, 0.1% NP-40, 10% glycerol, 1mM PMSF) then lysed in SDS Lysis Buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS). Chromatin was sheared to an average length of 300bp using a Palmer immersion sonicator (Three 1-minute rounds of 10s on/2s off at 40% amplitude) and diluted 1:10 in ChIP Dilution Buffer (50 mM Tris-HCl pH8.0, 5 mM EDTA, 200 mM NaCl, 0.5% NP-40, 1 mM PMSF). The samples were pre-cleared with 50µL of Protein A Dynabeads (ThermoFisher Scientific) at 4°C for 3 hours, then Input samples were set aside and stored at -80°C. Next, 10µL of the appropriate antiserum was added (anti-Pbx4 or anti-NF-YA) and the samples were incubated rotating at 4°C overnight. The immune complexes were precipitated onto 50µL of Protein A Dynabeads, which were washed five times with Wash Buffer (20 mM Tris-HCl pH8.0, 2 mM EDTA, 500 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF), three times with LiCl Buffer (20 mM Tris-HCl pH8.0, 2 mM EDTA, 500 mM LiCl, 1% NP-40, 0.1% SDS, 1 mM PMSF), and three times with TE Buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 1 mM PMSF). To elute chromatin, the beads were incubated in 50µL of fresh Elution Buffer with shaking at 1,500 RPM for 15 minutes at 25°C then 15 minutes at 65°C. To reverse crosslinks, 2µL of 5M sodium chloride was added to the samples, which were then incubated at 65°C overnight. Amin et al. 2015 doi:10.1016/j.devcel.2014.12.024.
Experimental Factor: immunoprecipitate
anti-Pbx4