Illumina HiSeq 2500 paired end sequencing; ChIP-seq for Prep on whole zebrafish embryos at 3.5 and 12hpf
ChIP-seq for Prep on whole zebrafish embryos at 3.5 and 12hpf
Groups of 500 zebrafish embryos (total of 10,000 at 3.5hpf and 5,000 at 12hpf per biological replicate) were dissociated in 1XPBS by pipetting and fixed for 10 minutes in 1% formaldehyde. Fixation was stopped by the addition of glycin to a final concentration of 125mM and cells were pelleted and frozen in liquid nitrogen.  DNA fragments were recovered in 10μl nuclease free water using a PCR purification mini-elute kit (Qiagen). Cell pellets were processed following a ChIP protocol described previously (Amin et al., 2015). Nuclei were extracted by the addition of 500μl L1 buffer (50mM Tris-HCl pH8.0, 2mM EDTA, 0.1% NP-40, 10% glycerol, 1mM PMSF) followed by incubation for 5 minutes on ice and pelleted by centrifugation (3000rpm, 5 minutes at 8°C). Nuclei were lysed in 300μl SDS lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA, 1% SDS) and chromatin sheared into smaller fragments (300bp on average) by 3 rounds of sonication with a Palmer sonicator (10 seconds ON – 2 seconds OFF for a total of 1 minute per round, amplitude 40%).  Samples were diluted 10 times in dilution buffer (50mM Tris-HCl pH8.0, 5mM EDTA, 200mM NaCl, 0.5% NP-40, 1mM PMSF) and pre-cleared by the addition of 50μl protein-A dynabeads (ThermoFisher Scientific) and incubation for 3 hours at 4°C. After removal of the beads, 10ul of anti-Prep or pre-bleed antiserum was added (see Key Resources Table for a full list of antibodies used in this study). Immune complexes were precipitated by the addition of 50μl of protein-A dynabeads (ThermoFisher Scientific) and incubated for 3 hours at 4°C. Beads were washed 5 times in wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA, 500mM NaCl, 1% NP-40, 0.1% SDS, 1mM PMSF), 3 times in LiCl buffer (20mM Tris-HCl pH8.0, 2mM EDTA, 500mM LiCl, 1% NP-40, 0.1% SDS, 1mM PMSF) and 3 times in TE buffer (10mM Tris-HCl pH8.0, 1mM EDTA, 1mM PMSF).  Chromatin fragments were eluted by the addition of 50μl of freshly made elution buffer (10mM Tris-HCl pH8.0, 1mM EDTA, 2% SDS) and incubation at 25°C for 15 minutes followed by an incubation at 65°C for another 15 minutes. Then, DNA fragments were reverse cross-linked by adding 2.5μl of 5M NaCl and incubating at 65°C O/N. Finally, DNA fragments were recovered in 10μl nuclease free water using a PCR purification mini-elute kit (Qiagen).   ChIP DNA fragments and their corresponding Input were quantified on a Qubit with the dsDNA HS assay kit (ThermoFisher Scientific). 10ng of DNA was used for library preparation using the Tru-seq ChIP Sample Preparation Guide (Illumina Inc). For samples containing less than 10ng of DNA the entire eluted DNA was used. Briefly, sample DNA was blunt-ended and phosphorylated, and a single 'A' nucleotide added to the 3' ends of the fragments in preparation for ligation to an adapter with a single-base 'T' overhang. Omitting the size selection step, the ligation products were then PCR-amplified to enrich for fragments with adapters on both ends. Libraries were sequenced on an Illumina HiSeq2500 Sequencer.  Amin, S., Donaldson, I.J., Zannino, D.A., Hensman, J., Rattray, M., Losa, M., Spitz, F., Ladam, F., Sagerstrom, C., Bobola, N., 2015. Hoxa2 selectively enhances Meis binding to change a branchial arch ground state. Dev. Cell 32, 265–277. doi:10.1016/j.devcel.2014.12.024
Experimental Factor: age
12
Experimental Factor: immunoprecipitate
anti-Prep