NextSeq 500 paired end sequencing; Time course DNase assays on Drosophila melanogaster and Drosophila virilis embryos during embryogenesis II
Time course DNase assays on Drosophila melanogaster and Drosophila virilis embryos during embryogenesis II
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.  1g of frozen embryos were dounce-homogenized, 20 times with a loose pestle and 10 times with a tight pestle, in 10 ml of chilled HB buffer (15 mM Tris-HCl (pH 7.4), 0.34 M sucrose, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA and 0.2 mM EGTA) on ice. The lysate was filtered through 2 layers of Miracloth and spun at 3,500g for 10 min to pellet the nuclei. The nuclei were then washed in 10 ml of chilled HB buffer, spun at 3,500g for 10 min and resuspended in 3 ml of PBTB buffer (5% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100 in PBS). The nuclei were dissociated by passing them ten times through a 20-G needle and then ten times through a 22-G needle using a 5-ml syringe, and filtered through a 20-micrometer Sefar Nitex membrane. The total number of nuclei was estimated by microscopy. DNase treatments were based closely on PMID: 23821440. Briefly, nuclei were permeabilised for 30min at 4C in PBS with 0.1% Triton-X and 0.02% NP40Nuclei were spun down at 2000G and resuspended in R-buffer [7.5mM Tris pH8, 45mM NaCl, 30mM KCl, 6mM MgCl2, 1mM CaCl2). Nuclei [~20x10e6 per digest] were then digested in 500uL R-buffer at 37C for 3min in a range of concentrations of DNase enzyme (10, 20, 30 or 50 units). After three minutes, the reaction was stopped with 500uL Stop-buffer [50mM Tris, pH-8, 100mM NaCL, 0.1% SDS, 100mM EDTA pH-8]. Reactions were then heated at 55C for 2 hours with 25uL of ProteinaseK [25mg/mL] and 300microg of RNaseA. An additional 25uL of ProK was then added and the reaction incubated over night at 65C. Following this step, digested DNA was isolated using Qiagen isolation kits and run out on a 1% agarose gel to assess digestion levels (as in PMID: 23821440). Optimal digests were then fractionated using sucrose gradients as in PMID: 23821440 to generate 330uL fractions. Fracitons 2 and 3 were then pooled and tested for quality using qPCR for known control regions of open chromatin. The digests with the highest qPCR enrichment were selected for library preparation.  Illumina libraries were prepared according to manufacturers recommendations with small modifications.  In short, 3-10ng of RNase treated, and reverse cross-linked DNA from our DNase protocol was end-repaired and terminal adenosine residues were added using the NEBNext reagents.  Modified versions of the Illumina PE adapters containing sample-specific molecular barcodes were ligated, after which the material was size selected with a mean of ~300 bp (size equals sheered chromatin fragments plus adapters) using AMPure beads.  PCR amplification was performed using PE1.0 and PE2.0 primers (Illumina) for 16 cycles according to manufacturer's recommendation using the Phusion High-Fidelity PCR Kit (Finnzyme).  The PCR-amplified library was purified using AMPure beads.