title: GSM6045753: Trachea_8807_INTACT_Lola_I_14-17h_2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Nuclei were isolated from INTACT embryos 14-17h AED by resuspending and douncing embryos in HBS buffer (0.125 M Sucrose, 15 mM Tris (pH 7.5), 15 mM NaCl, 40 mM KCl, 2 mM EDTA, 0.5 mM EGTA). The nuclei were further dissociated using syringe (22.5 gauge needle) 10 times. After washins with HBS buffer, nuclei were incubated with Dynabeads® M-280 Streptavidin beads( Invitrogen, # 11205D) for 30 minutes with end to end rotation at 4°C. A magnet was used to separate the bead bound nuclei, and the beads were washed thoroughly with HBS buffer. Purity of nuclei isolation was determined by staining nuclei with DAPI, and presence of free nuclei, unbound to beads, were used as indicators of contamination. Isolated nuclei were used for ChIP-seq. ChIP-seq was performed as follows. ~100 mg embryos were used per ChIP, and 5 µg chromatin was used for tissue-specific ChIP-seq experiments. Fixed embryos were homogenized by douncing in an ice cold A1 buffer (15 mM HEPES (pH 7.5), 15 mM NaCl, 60 mM KCl, 4 mM MgCl, 0.5% Triton X-100, 0.5mM DTT, protease inhibitors) and A2 buffer (15 mM HEPES (pH 7.5), 140 mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, protease inhibitors) in a tissue grinder for 10-15 times in A1 and A2 buffer each. Then the sonication of the chromatin was performed with a Bioruptor Pico for four-five rounds of 30 s on and 30 s off cycles. The sonicated chromatin was cleared by centrifugation and the supernatant was used for ChIP. Chromatin was incubated with antibodies pre-bound to Dynal magnetic beads (IgA or IgG) overnight with end-to-end rotation at 4°C and washed with an ice cold RIPA buffer (50 mM HEPES (pH 7.5), 1mM EDTA, 0.7% sodium deoxycholate, 1% NP-40 (IGEPAL CA-630), 0.5M LiCl). Eluted, reverse cross-linked DNA was then purified using phenol-chloroform-isoamylalcohol phase separation and ethanol precipitation. ChIP-seq libraries were prepared from 5-15 ng ChIP DNA or 100 ng input DNA according to the manufacturer's instructions (NEBNext ChIP-Seq Library Prep kit).