title: NextSeq 500 paired end sequencing; Hi-C in Drosophila embryos at NC14 depleted of Beaf-32, CP190 or CTCF using the Bridge Adaptor Protocol
design: Hi-C in Drosophila embryos at NC14 depleted of Beaf-32, CP190 or CTCF using the Bridge Adaptor Protocol
library_name: CTCF_KO_2-3h_bridge22_rep1_p
library_construction_protocol: Adult flies from specific genotypes were placed in medium-sized cages. After three 1 h pre-lays, a 1 h embryo laying was allowed on apple juice agar plates with yeast paste. 2 hours later, embryos were immediately dechorionated using 50% bleach and formaldehyde fixed in (1:3) cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and n-heptane on a shaker table at RT for 15 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 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).  5-15 million nuclei extracted from embryos of specific genotypes were permeabilised with SDS, and then incubated with the restriction enzyme DpnII overnight. The restriction enzyme was inactivated, and then digested DNA ends were ligated to a biotinylated bridge oligo. After washes, the bridge oligos were phosphorylated, and ligated to each other. Then, chromatin was extracted, de-crosslinked, and DNA was purified and RNAse-treated. Purified DNA was sheared to 200-400bp and size-selected using beads. Finally, biotinylated DNA was pulled-down with streptavidin beads, and used for NGS library preparation.   Hi-C libraries were prepared using the kits SWIFT Accel-NGS 2S Plus DNA Library Kit for Illumina (biocat 21024-SWI) and SWIFT 2S Indexing Kit for Illumina (biocat 26148-SWI)