title: GSM6045788: Dme_emb_orc4_14-17h_Rpb3_3; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: ChIP-seq were 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).