Illumina HiSeq 2000 paired end sequencing; Tissue-specific DNase-seq during Drosophila melanogaster embryogenesis
Tissue-specific DNase-seq during Drosophila melanogaster embryogenesis
Transgenic Drosophila melanogaster carrying a mesodermally expressed tagged histone marker (1) were grown in population cages at 25 degr.   Staged 2 hr collections of embryos were made on apple-agar plates and aged at 25 degr until the required stage of development.   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, 1.8 % 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, snap frozen in liquid nitrogen, and stored at -80 degr. A small number of embryos from each collection was set aside for staging.    1.Bonn, S., Zinzen, R.P., Perez-Gonzalez, A., Riddell, A., Gavin, A.C., and Furlong,  E.E. (2012). Cell type-specific chromatin immunoprecipitation from multicellular  complex samples using BiTS-ChIP. Nat Protoc 7, 978-994.   Nuclei were isolated from fixed, frozen embryos as described in (1). Nuclear pellets were resuspended in PBS with 5% BSA, 0.1% TritonX-100 and 0.2% Igepal-630 and rotated at 4 degr for 30 mins.  Primary antibody staining was performed overnight at 4 degr in 3mL PBTB (PBS with 5% BSA and 0.1% TritonX-100) per 1g frozen embryos.  Secondary antibody staining was performed for 1 hr at 4 degr in PBTB. Following each antibody staining nuclei were washed twice consisting of pelleting and resuspension in 10mL PBTB. An aliquot of stained, unsorted nuclei was put aside to represent the whole-embryo. FACS was performed to isolate the desired populations, and nuclei were consolidated and pelleted. For DNase digestion, nuclei were resuspended in R-buffer [7.5mM Tris pH8, 45mM NaCl, 30mM KCl, 6mM MgCl2, 1mM CaCl2). Nuclei [4-20x10e6 per digest] were digested in 500uL R-buffer containing 2-20 units of DNaseI depending on nuclei number (where possible with multiple concentrations) at 37C for 3min, and the reaction was stopped by adding 500uL Stop-buffer [50mM Tris, pH-8, 100mM NaCL, 0.1% SDS, 100mM EDTA pH-8]. A small control digest without DNaseI was performed to assess DNA integrity. Following addition of RNaseA, samples were incubated at 55 degr for 10 mins, then 25uL of ProteinaseK [25mg/mL] was added and the samples were incubated overnight at 65 degr to reverse cross-links. A small aliquot was run on a 1% agarose gel to assess digestion levels, and optimal digests were size fractionated using sucrose gradients. DNA fragments ~100-500bp were isolated from fractions using a Qiagen PCR clean up kit and checked for enrichment in known hypersensitive sites by qPCR. The digests with the highest qPCR enrichment were selected for library preparation.    (1). Bonn, S., Zinzen, R.P., Perez-Gonzalez, A., Riddell, A., Gavin, A.C., and Furlong,  E.E. (2012). Cell type-specific chromatin immunoprecipitation from multicellular  complex samples using BiTS-ChIP. Nat Protoc 7, 978-994.   Illumina-compatible DNA sequencing libraries including sample-specific (index read) and molecular barcodes (in-line) were prepared using the NextFlex qRNA-seq Kit v2 kit (Biooscientific #NOVA-5130-12) largely according to manufacturer's instructions. In short, between ~1 and 15ng DNA consisting of ~100-500bp fragments that result from DNase digestion was end-repaired and terminal adenosine residues were added. Adapters containing in-line molecular barcodes were ligated, after which the material was size selected using AMPure beads (negative selection with 0.6X beads, then postive selection with 0.98X beads). PCR amplification was performed using barcoded primers to introduce sample barcodes for 12-18 cycles, depending on input amount. The PCR-amplified library was purified using AMPure beads, quantified using a Qubit High-sensitivity DNA kit, and sized on an Agilent Bioanalyzer chip.