title: GSM2092686: Nuc_OP27_WT_147bp_N3; Mus musculus; MNase-Seq
library_construction_protocol: 2.5x10^6 of wild-type OP27 cells or OP27 cells stably transduced to express an Evi1-directed shRNA were seeded. Mono-nucleosomal DNA was prepared essentially as described in (Rizzo et al., 2012). In brief, chromatin was crosslinked by adding methanol-free 16% formaldehyde solution (Pierce) to a final volume of 1% to the culture medium. Crosslinking was quenched with 1.25 mM Glycine, cells were washed twice with PBS containing protease inhibitors (PBS/PI, Roche), and scraped into 1.5 ml reaction vessels. Cells were then washed once with PBS/PI. Nuclear extracts were prepared and the protein concentration was measured using Bradford assay. 100 mg of total nuclear extract were then digested for 1 hour at 37°C with a dilution series of micrococcal nuclease (1 to 40 units/ml, Takara) and 10 µl of digested material was analyzed on a 2% agarose gel to identify equally digested samples showing only a signal at the size on mono-nucleosomes (~147bp). Equally digested samples were again loaded on a 2% agarose gel and the area of the gel comprising DNA of a size between 50 and 400bp was analyzed by densitometry using Quantity one software (Biorad). Density values for each sample and lane were extracted and subjected to a Pearson correlation analysis using R (www.r-project.org). Only samples with correlation coefficients >0.9 for each sample comparison were used for library preparation and MNase-seq analysis. Sequencing libraries were generated according to Illumina’s Low Throughput TruSeq library preparation protocol with minor modifications: 200 ng of mono-nucleosomal DNA as quantified using Quant-iT Picogreen assay (Life Technologies) were used as starting material. The first AmpPure bead-cleanup step (after End Repair and before A-Tailing reactions) was replaced with a MinElute purification step (elute with 30 μl elution buffer) to remove the size-selection bias of bead-cleanups, which favors larger (>200 bp) DNA fragments. After the column cleanup and 3’ adenylation, 1:5 diluted adapters were used for ligation reactions. Finally, when enriching DNA fragments, listed PCR conditions were followed, but only 1μl of ligation product was used as input to ensure the PCR primers remain in excess and to avoid bubble amplification products. Sequencing was carried out using an Illumina HiSeq 2500.