title: GSM2266319: MNase-Seq GST RNAi replicate 1; Drosophila melanogaster; MNase-Seq
library_construction_protocol: After 6 days cells were harvested and 1/10 volume of fixing solution (1 M NaCl, 0.5 M HEPES pH 8, 10 mM EDTA, 5 mM EGTA, 11% FA) directly added to the medium. After an incubation of 10 min at 18°C, glycine was added to 125 mM and cells collected by centrifugation. To isolate the nuclei, cells were washed in 10 ml of cold PBS and resuspended in 1 ml PBS (containing protease inhibitor cocktail (PIC, Roche) and 0.5% Triton X-100) per 1 x 108 cells and rotated for 15 min at 4°C. Nuclei were then washed in 10 ml PBS and resuspended in 1 ml RIPA buffer (incl. PIC) per 1 x 108 cells and CaCl2 was added to 2 mM f.c.. Aliquots of 600 µl were treated with 1.2 U of MNase for 20 min at 37°C, then 13 µl of 0.5 M EGTA and 400 µl RIPA were added. The chromatin was sheared in a Covaris sonicator in a 12x12 fiber tube at 50 W, 20% output for 8 min. After a centrifugation at 17,000 x g for 20 min, the supernatant was used for subsequent H3 ChIP. Recovered DNA was quantified using the Qubit® dsDNA HS Assay Kit (Life Technologies, Cat.no.Q32851) and sequencing libraries were prepared using the MicroPlex Library Preparation kit (Diagenode, Cat. No. C05010011) starting with 2 ng DNA whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles).
attributes.tag: GEO Accession
attributes.value: GSM2266319