Name: L16_Dvir_Bin-EHUD-14-17h-IP-64-9
Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: "Wildtype Drosophila virilis (white eye mutation line) flies were grown in population cages at 25 degr. Staged populations of embryos were collected and aged at 25 degrees till the required stage of development is reached. Two independent collections were performed for each timepoint. 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, 485ul of 37% Formaldehyde, pH 8.0) and 30ml 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." "30 ug of chromatin was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF, 1ul Leupeptin/Pepstain and 1 ul Aprotinin). For precipitation, 3ul Dvir-anti Bin (EHUD) antibody was added to each aliquot and incubated overnight at 4 degres on a rotating shaker. For each precipitation, 15 or 20 ul of 50% Protein A Sepharose (PAS) suspension were washed once with 1mL RIPA buffer + 1mg/ml BSA for blocking. In the morning, The antibody/antigen complexes were precipitated with 15 or 20 ul protein A sepharose at 4 degres for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with increase NaCl (500 mM), 1x in 250mM LiCl / 10mM Tris-HCl pH 8,0 / 1mM EDTA / 0,5% NP-40 / 0,5% sodium deoxycholate and 2x in TE. The DNA was eluted after protease digest and RNAase treatment by incubation at 65 degr overnight and phenol chloroform extracted. " "~1g of embryos of early time points (or ~0.5 from later time points) were thawn quickly and resuspended in 15ml ice-cold PBS/Triton + protease inhibitors. Dounce homogenization 20x with loose pestle on ice followed by centrifugation at 400g for 1min at 4 degrees and transfer SN to fresh tube. Centrifuge SN at 1100g for 10 min at 4 degrees and discard SN. Resuspend pellet in 15 ml ice-cold Cell lysis buffer + protease inhibitor Dounce 20x with tight pestle on ice. Transfer two equal aliquots into 15 ml falcon tubes. Centrifuge at 2000g for 4min at 4 degrees to pellet the nuclei. Discard SN. Resuspend pellet in 1ml of ice-cold Nulear-Lysis Buffer + proteas inhibitor and incubate for 20 min at RT Add 1ml ice-cold Nuclear Lysis Buffer + proteas inhibitors Sonicate using Bioruptor in 15 ml faclon tubes with a max of 2 ml in them for 18 cycles with 30 seconds ON / 30 seconds OFF. Transfer to 1.5 ml tubes and centrifuge at 14000 rpm for 10 min at 4 degrees Take SN=chromatin and make aliquots of 400 ul and flash freeze in liquid nitrogen and store at -80 degrees. Cell lysis buffer: 5mM Hepes pH 8, 85mM KCl, 0.5% NP40 + protease inhibitors Nuclear lysis buffer: 50mM Hepes pH 8, 10mM EDTA Na2, 0.5% N-Laurylsarkosin + protease inhibitors " "Solexa libraries were prepared according to manufacturers recommendations with small modifications. In short, 1-10ng of IP-purified, RNase treated, and reverse cross-linked genomic DNA was end-repaired and terminal adenosine residues were added using the NEBNext reagents. Indexed adapters (in house) were ligated, after which the material was size selected at ~230-250 bp (size equals sheered chromatin fragments plus adapters) on a 2% GelGreen (Biotium) stained agarose gel using SafeXtractor-100 gel extractors (5Prime) under blue light. PCR amplification was performed using PE1.0 and PE2.0 primers (Illumina) for 14 cycles for Input samples and 16 cycles for IP-ed samples using the Phusion High-Fidelity PCR Kit (Finnzyme). The PCR-amplified library was purified on a 2% agarose gel, avoiding unincorporated primers and primer-self-ligation products. Library quality was assessed on a Bioanalyzer 2100 system (Agilent). "