title: NextSeq 500 paired end sequencing; Chromatin immunoprecipitation of Drosophila embryos at 6-8h AEL to identify Beaf-32, CP190, CTCF and SuHw proteins binding
design: Chromatin immunoprecipitation of Drosophila embryos at 6-8h AEL to identify Beaf-32, CP190, CTCF and SuHw proteins binding
library_name: BEAF_68h_WT_rep1_p
library_construction_protocol: Wildtype Drosophila melanogaster (Oregon R) flies 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 10 ml 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 ChIPs, 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. Chromatin 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 lyzed in 50mM Hepes, pH 8, 10mM EDTA?Na2, 0.5% N-Laurylsarkosin + protease inhibitors at RT for 20 min and the chromatin was sheared to ca. 250 bp fragments using a Bioruptor sonicator waterbath (Diagenode). After clearing the chromatin by centrifugation (20.000g at 4 degr for 10 min), aliquots were frozen at -80 degr.   Chromation IP Step:  5 micrograms of chromatin were mixed with TE and cold IP dilution buffer. 1% Input was isolated at this step. The chromatin was incubated with the appropriate antibody overnight on a rotating wheel. Then, the antibody/antigen complexes were incubated with Protein A/G Beads, followed by multiple washes. The DNA was eluted after RNAase treatment, protease digestion and phenol chloroform extraction.    For ChIP NGS library prep the following kit was used: NEBNext Ultra II DNA Library Prep Kit for Illumina E7645S