NextSeq 500 paired end sequencing; CUTnTag against Beaf-32, CP190 and CTCF in Drosophila melanogaster wild-type and insulator-depleted embryos at stage NC14
CUTnTag against Beaf-32, CP190 and CTCF in Drosophila melanogaster wild-type and insulator-depleted embryos at stage NC14
Wildtype Drosophila melanogaster (yellow white - yw) flies or crosses for insulator depletions were grown in population cages at 25 degr. Independent collections of staged embryos were performed for each biological replicate. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 700 ul cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30ml n-heptane on a shaker table at RT for 15 minutes. Specifically for CP190 C&Ts, the same collection procedure was used, but using a final 3.0 % concentration of formaldehyde, for 30 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collections was set aside to stage each collection. Nuclei Preparation Step: Nuclei were extracted from the frozen embryos by Dounce homogenization and differential centrifugation (Cell lysis buffer: 5mM Hepes, pH 8, 85mM KCl, 0.5% NP40 + protease inhibitors). The nuclei were counted at a flow cytometer, divided in 50K aliquots and snap-frozen in liquid nitrogen. Antibody Binding and Tagmentation Step: 50K nuclei from D. Melanogaster and D. Virilis were thawed and mixed together on ice. ConA beads were then added to the nuclei mixture. The nuclei were permeabilized, and then the nuclei-beads complex was incubated with the primary antibody and slowly rotated (5 rpm) overnight at 4C. A secondary antibody solution (1:100, 100 _l/sample) was added to the beads and incubated for 1h at room temperature. Samples were then washed three times and incubated with a pA-Tn5-oligo adapter complex. Tagmentation allowed to happen at 37C for 1h. Samples were de-crosslinked, digested with protease, phenol/chloroform - extracted, column purified and RNase treated. C&T NGS libraries were prepared using P5 and P7 primers and the NEBNextï¿½ High-Fidelity 2x PCR Mater Mix (M0541L).