Two technical replicate libraries were constructed for

Two technical replicate libraries were constructed for selleck chemical 17-AAG each DNA-Seq sample. Two libraries were prepared from two biological replicates of each RNA material (RNAi or mock treated). RNAi dsRNA for RNAi treatment [38] was produced by in vitro transcription of a PCR generated DNA template from Drosophila genomic DNA containing the T7 promoter sequence on both ends. Target sequences were scanned to exclude any complete 19 mer homology to other genes [39]. The dsRNAs were generated using the MEGAscript T7 kit (Ambion, Austin, TX) and purified using RNAeasy kit (Qiagen, Valencia, CA). Two different primer sets were used for each target gene, and the one with better RNAi efficiency was used for downstream experiments.

The selected primer sequences for generation of msl2 dsRNA template by PCR were as follows: forward, 5��-taatacgactcactatagggTTGCTCCGACTTCAAGACCT-3��, and reverse, 5��-taatacgactcactatagggGCATCACGTAGGAGACAGCA-3�� and the selected primer sequences for generation of mof dsRNA template were as follows: forward, 5��-taatacgactcactatagggGACGGTCATCACAACAGGTG-3��, and reverse, 5��-taatacgactcactatagggTGCGGTCGCTGTAGTCATAG-3��. For RNAi treatment, S2 cells were resuspended in serum free media at 2��106 cells/ml. Twenty ��g dsRNA was added to 1 ml of cell suspension and incubated for 45 min at room temperature. Cells with the same serum free media treatment but without added dsRNA were used as mock treated controls. After the incubation, 3 ml complete medium was added and the cells were cultured for another 4 d.

Cells were collected and split into three aliquots for mRNA extraction, chromatin immunoprecipitation, and western analysis. ChIP For ChIP [40], 5�C10��106 S2 cells were fixed with 1% formaldehyde in tissue culture media for 10 min at room temperature. Glycine was added to a final concentration of 0.125 M to stop cross-linking. After 5 min of additional incubation and two washes with ice-cold PBS, cells were collected and resuspended in cell lysis buffer (5 mM PH 8.0 PIPES buffer, 85 mM KCl, 0.5% Nonidet P40, and protease inhibitors cocktail from Roche, Basel, Switzerland) for 10 min and then resuspended in nuclei lysis buffer (50 mM PH 8.1 Tris.HCl, 10 mM EDTA, 1% SDS and protease inhibitors) for 20 min at 4��C. The nuclear extract was sheared to 200�C1,000 bp by sonication on ice for 8 min (pulsed 8 times for 30 s with 30 s intervals using a Misonix Sonicator 3000; Misonix, Inc.

Farmingdale, NY). The chromatin solution was then clarified Cilengitide by centrifugation at 14,000 rpm for 10 min at 4��C. Five ul anti-H4AcK16 (Millipore, Billerica, MA) was incubated with the chromatin for 2 h and then was bound to protein A agarose beads at 4��C overnight. The beads were washed three times with 0.1% SDS, 1% Trition, 2 mM EDTA, 20 mM PH 8.0 Tris, and 150 mM NaCl; three times with 0.1% SDS, 1% Trition, 2 mM EDTA, 20 mM PH 8.0 Tris, and 500 mM NaCl; and twice with 10 mM PH 8.

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