Genome-wide mapping of matrix attachment regions in Drosophila melanogaster
Nuclei were isolated from 1 gm of 0-16 hrs Drosophila embryos.  To prepare nuclear matrix, chromatin was removed by DNaseI digestion in a buffer containing 20 mM Tris pH 7.4, 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 0.125 mM spermidine 1 mM PMSF, 0.5% Triton-X 100, and 200 µg/ml DNaseI (Sigma) at 4°C for 1 hr. Chromatin depleted nuclei were extracted with 0.4 M NaCl for 5 min in extraction buffer containing 10 mM Hepes pH 7.5, 4 mM EDTA, 0.25 mM spermidine, 0.1 mM PMSF, 0.5% (v/v) Triton X-100 and another 5 min with 2 M NaCl in the extraction buffer.  The final pellet after extraction was washed twice with wash buffer (5 mM Tris pH 7.4, 20 mM KCl, 1 mM EDTA, 0.25 mM spermidine, 0.1 mm PMSF).  To remove RNA, RNaseA was added to a final concentration of 20 µg/ml. Incubation was carried out at 37°C for 30 min to remove all associated RNA. This was followed by digestion with 100 µg/ml Proteinase K at 55°C for 1 hr. Matrix attached regions (MAR_DNA) was recovered by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitation. Libraries were prepared according to manufacturer’s instruction using Fragment Library Construction Kit from Applied Biosystems (Life Technologies). Libraries were prepared from two independent biological replicates of 0-16 hrs Drosophila embryos. Briefly, 5 µg of DNA was end-repaired, ligated to P1 and P2 adaptors, and size selected on a gel where 50-100 bp fragments were selected. Using the adaptors, the size-selected DNA was amplified for 3 cycles to generate the fragment library. Library was quantified using the Agilent 2100 Bioanalyser (Agilent Technologies Inc.). The libraries were sequenced on ABI SOLiD v2 Sequencer according to the manufacturer's instruction.