50 million S2 cells were harvested and washed with PBS. Cells were counted very carefully to ensure that the same number of cells was collected for each sample. The cells were resuspended in 1 ml of crosslinking solution (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and 37% formaldehyde was added to a final concentration of 1%. The tube was rotated at room temperature for 10 minutes and the reaction was quenched with 2.5 M glycine at a final concentration of 125 mM. The cells were spun down immediately after addition of glycine and the solution was replaced with fresh PBS that contained 125 mM glycine. The cells were resuspended and rotated at room temperature for 5 minutes. The cells were pelleted into equal aliquots by centrifugation with 2000 rpm for 5 minutes.  Cells were permeabilized with NP-40. The chromatin was digested with various amount of MNase at 25 ºC for 10 minutes. Digested chromatin was then analyzed on an agarose gel to reveal the digestion pattern. We aimed to achieve a slightly under-digested condition with di- and tri-nucleosomes still visible. In addition, the digestion pattern of each sample needed to be exactly the same. After the optimal amount of MNase was determined for each individual experiment, a new aliquot of the cells was again digested with MNase at 25 ºC for 10 minutes. The reaction was stopped by addition of EDTA, NaCl and SDS to an end 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 65 ºC overnight in TE buffer. The sample was treated with RNaseA (0.2 mg/ml) for 30 minutes at 37 ºC, and with Proteinase K (0.05 mg/ml) for 2 hours at 50 ºC. Finally, DNA was purified using Minelute columns (Qiagen) and eluted in nuclease-free water. The eluted DNA was then analyzed 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 Accession
GSM3344611