Illumina HiSeq 2000 sequencing; Time course Chromatin Immunoprecipitation on Drosophila virilis embryos during embryogenesis
Time course Chromatin Immunoprecipitation on Drosophila virilis embryos during embryogenesis
"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." "Between 30 and 70 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 Mef2 (DRE5 or DRC4) antibody was added to  each aliquot and incubated overnight at 4 degres on a rotating shaker.  For each precipitation, 20 or 25 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 20 or 25 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).  "