title: GSM1694841: Dm_S2_cells_18C_MNase_HIGH_rep1; Drosophila melanogaster; MNase-Seq
library_construction_protocol: 1) Differential MNase-seq:  MNase digestion of chromatin of Drosophila embryos was performed as described (Petesch SJ and Lis JT, Cell 2008, 134:74-84) with few modifications. In brief, 2 g of embryos collected overnight were homogenized in 5 ml of buffer A1 (60mM KCl, 15 mM NaCl2, 4mM MgCl2, 15 mM HEPES pH7.6, 0.5 mM DTT, 0.5% Triton X-100, protease inhibitors) containing 2% of formaldehyde and crosslinked for 15’ at room temperature. Reaction was stopped by adding 1M of glycine to a final concentration of 225 mM followed by 5’ incubation. Cross-linked nuclei were filtered through Miracloth (475855, EMD Millipore), loaded on top of the sucrose cushion (0.3 M sucrose in buffer A1) and centrifuged at 700 g, 4°C for 5’. Nuclei were washed 2X in buffer A1, 2X in buffer D (25% glycerol, 5 mM Mg Acetate, 50 mM Tris pH 8.0, 0.1 mM EDTA, 5 mM DTT) and re-suspended in 1 ml of buffer MN (60 mM KCl, 15 mM NaCl, 15 mM Tris pH 7.4, 0.5 mM DTT, 0.25 M sucrose, 1.0 mM CaCl2).    Micrococcal nuclease (MNase) digestion of chromatin was performed by the addition of 10 (MNase-LOW) to 250 (MNase-HIGH) total units of MNase (70196Y, Affymetrix) to 200 mkl of isolated nuclei. Following 10’ of incubation at 25°C, the reactions were stopped with EDTA and SDS added to final concentrations of 12.5 mM and 0.5% respectively. MNase digested DNA was decrosslinked by incubation in elution buffer containing 1% SDS, 0.5 mg/ml Proteinase K, 0.1 M NaHCO3 for 2 h at 37°C and 16 h at 65°C, purified with QIAquick PCR Purification kit (28106, QIAgen) and analysed on 2% agarose gel. DNA fragments corresponding to mono-nucleosome (~147 bp) were excised from the gel and purified for further analysis.    A similar protocol was used for MNase digestion of chromatin from 2 ×10^8 Drosophila S2 cells. For S2 cells, DNA fragments corresponding to mono- (~147 bp) and di- (~300-350 bp) nucleosomes were gel-isolated.    2) Differential MNase-ChIP-seq:  For MNase-HIGH-ChIP-seq, 1 ml of nuclei was prepared from 2 g of Drosophila embryos as described above followed by the complete digestion of chromatin to mono-nucleosomes with 250 U of MNase per 200 mkl of nuclei for 10’ at 25°C. After that, nuclei were lysed immediately for 10’ at 4°C by the addition of equal volume of 2X ChIP lysis buffer (2% SDS, 20 mM EDTA, 100 mM Tris-HCl pH 8.1, 0.2 mM PMSF) and stored at -80°C before use.    For MNase-LOW-ChIP-seq, 12 ml of nuclei was prepared from 50 g of Drosophila embryos followed by the digestion of chromatin with 10 U of MNase per 200 μl of nuclei for 10’ at 25°C. After the reaction was stopped, nuclei were collected by centrifugation at 9000 g for 10’ at 4°C and lysed for 1 hour at 4°C in 6 ml of a buffer containing 0.5 M NaCl, 10 mM HEPES pH 7.6, 1 mM EDTA and 0.2 mM of protease inhibitor PMSF. Lysed nuclei were spun at 15000 g for 15’ at 4°C and supernatant containing soluble chromatin was loaded onto 5-30% sucrose gradient prepared on Gradient Master (Biocomp). Molecular weight fractions were formed by centrifugation at 26000 rpm for 16 h at 4°C in SW 40 Ti swing rotor. 1ml fractions were collected and 200 μl aliquots were decrosslinked and analysed on 2% agarose gel. Fractions containing mono-nucleosomes were used for ChIP.    Chromatin immune-precipitations (ChIPs) were performed as described (Moshkin YM, et al., Mol Cell Biol 2012, 32:675-688). In brief, lysed nuclei from MNase-HIGH digestion or isolated mono-nucleosomes from MNase-LOW digestion experiments were diluted with 10 volumes of ChIP dilution buffer (1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20mM Tris-HCl pH 8.1, 0.2 mM PMSF), incubated with ChIP-grade antibodies against histone H3 (ab1791, Abcam) or H2B (Moshkin YM, et al., Mol Cell Biol 2012, 32:675-688) overnight at 4°C and precipitated with pre-blocked protein A agarose (16-157, EMD Millipore). Following extensive washes: 3 times with a buffer containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1 and 150 mM NaCl and once with the similar buffer containing 500 mM NaCl, DNA was decrosslinked and purified with QIAquick PCR Purification kit (28106, QIAgen). Libraries for Illumina sequencing were prepared from 10 ng of DNA by Service XS (Netherlands) with Illumina kits, or in-house with SureSelect kit (G9691A, Agilent) starting from the end-repair step. For the input controls, libraries from sonicated or MNase digested genomic DNA were prepared. Libraries’ quality was assessed on Bioanalyzer using DNA1000 kit (5067-1504, Agilent). Libraries were paired-end sequenced (2x25 bp or 2x50 bp) on Illumina HiSeq 2000.
attributes.tag: GEO Accession
attributes.value: GSM1694841