GSM1575685: MNase MSL3 RNAi A; Drosophila melanogaster; MNase-Seq
Cells were permeabilized with NP-40. The chromatin was digested with microcccal nuclease (MNase) at 25¼C for 10 minutes. Digested chromatin was then analysed on an agarose gel to reveal the digestion pattern. The digestion pattern of each sample was exactly the same. The MNase digestion was stopped by addition of EDTA, NaCl and SDS to a final concentration of 20 mM, 150 mM and 1% respectively. The samples were placed on ice during handling to ensure that the MNase digestion was halted completely. The chromatin was then de-crosslinked overnight at 65¼C overnight in TE buffer. The sample was treated with RNaseA (0.2 mg/ml) for 30 minutes at 37¼C, and with Proteinase K (0.05 mg/ml) for 2 hours at 50¼C. Finally, DNA was purified using Minelute columns (Qiagen) and eluted in nuclease-free water. The eluted DNA was then analysed with the E-Gel Pre-cast Agarose Gels system (Invitrogen). Bands at positions corresponding to 150 bp, 300 bp, 450 bp, represented mono- di and tri-nucleosomes, respectively. The 150 bp fragment was purified from the E-gel according to manufacturers specifications and used to generate the library for the Illumina paired-end sequencing. To minimize the effect of PCR artefacts, only 3 cycles of PCR were performed in the library generation procedure.
GEO
GSM1575685