NextSeq 550 sequencing; ChIP-seq of Drosophila embryos to investigate chromatin state during dorsal-ventral patterning
ChIP-seq of Drosophila embryos to investigate chromatin state during dorsal-ventral patterning
We adapted the fixation and sorting procedure described in (Blythe and Wieschaus, 2015) for in situ Hi-C (Rao et al., 2014). Following a pre-collection period of at least one hour, fly embryos were collected on yeasted 0.4% acetic acid agar plates or apple juice plates at 25 °C. After 1 hour of collection, the embryos on the plate were incubated at 25 °C for 2 hours. Embryos were dechorionated for 2 min in 2.6% sodium hypochlorite, rinsed with water, and transferred to vials containing 2mL of PBS, 0.5% Triton X-100 and 6mL of heptane. Cross-linking was initiated by adding 100 µl of 37% formaldehyde, followed by vigorous shaking. After 10 min, samples were spun at 500 g for 1 min and the upper heptane layer was removed. 15 min after the start of fixation, 5 mL of PBS, 0.5% Triton X-100, 125 mM glycine was added to the embryos, followed by vigorous shaking for 1 min. The embryos were rinsed 3 times with PBS, 0.5% Triton X-100. Frozen embryos were homogenized in sonication buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1 % Triton, 0.1 % sodium deoxy- cholate, 0.1 % SDS and protease inhibitor) using a Dounce homogenizer. The sample were spun at 4,000 x g for 5 min and the pellet containing the intact nuclei was resuspended in same buffer supplemented with 0.5% N-Lauroylsarcosine and SDS to final concentration of 0.5 %. The chromatin was sheared to fragment size in the range of 200–500 bp using a Bioruptor (Diagenode). The solubilized chromatin fraction was cleared by centrifugation and used for immunoprecipitation after diluting 5 times with sonication buffer. Immunoprecipitation with either 2 µg of H3K27ac (Abcam, ab4729) or 5 µg of H3K27me3 (Abcam, ab6002) antibody was carried out on chromatin corresponding to 20-25 µl of embryos at 4 °C overnight. Chromatin - antibody complexes were captured for at least 3 hr using a mix of Protein A and G Dynabeads (Invitrogen). The captured immunoprecipitated complex were washed 10 min each with sonication buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1 % Triton, 0.1 % sodium deoxyolate, 0.1 % SDS), WashA (as sonication buffer, but with 500 mM NaCl), WashB (20 mM Tris pH 8, 1 mM EDTA, 250 mM LiCl, 0.5 % NP-40, 0.5 % sodium deoxycholate) and TE. After the washes Dynabeads were resuspended in 100  TE supplemented with 20 /ml RNase A and incubated at 50 °C for 30 min. Cross-linking was reversed by adding Tris pH 8.0 and SDS to a final concentration of 50 mM and 0.1% respectively and heating at 68 °C for at least 4 hr. Protein digestion was carried out by Proteinase K treatment at 55 °C for 2 hr, followed by purifying ChIP DNA using ChIP DNA Clean & Concentrator kit (Zymo research #D5205). ChIP-seq libraries were prepared on the ChIP DNA eluted in 60  of DNA elution buffer, using the NEBNext Ultra II DNA Library Prep Kit (NEB).