GSM1892895: jhd2D nucleosome  rep3; Saccharomyces cerevisiae; MNase-Seq
Cells were crosslinked for 20 minutes by addition of formaldehyde to 1% final concentration in the media and quenched by the addition of 1M glycine to 0.1M final concentration. 33 x 10^7 cells were harvested by centrifugation and washed twice with Phosphate-Buffered Saline and once with spheroplasting buffer (1M Sorbitol, 10 mM Tris.Cl pH 7.5, 15 mM 2-mercaptoethanol and protease inhibitors (PIs): 1 mM PMSF, 1μg/ml aprotinin, 1μg/ml leupeptin, 1μg/ml pepstatin A). Cross-linked cells were resuspended in 1ml fresh spheroplasting buffer before adding Lyticase (500 units; Sigma) and incubated in a 37oC water-bath shaker for 15-20 min with gentle agitation. Spheroplasts were washed twice with 25ml wash buffer (50mM Tris pH 7.5, 100mM KCl, 2.5mM MgCl2, 400mM Sorbitol, and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100mM NaCl, 5mM MgCl2, 1mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37oC for 15 min. Digestion was stopped by adding 100μL 10X stop buffer (50mM Tris.Cl pH 7.5, 500mM NaCl, 100mM EDTA, 20mM EGTA, 10% Triton X-100 and 1% Sodium Deoxycholate). After centrifugation, soluble chromatin (500μL) was mixed with an equal amount of elution solution (1% SDS, 100 mM sodium bicarbonate) and NaCl (200mM final concentration), reverse cross-linked for 5h at 65oC and ethanol precipitated overnight at -20oC. After centrifugation, the DNA pellet was washed with cold 70% ethanol, air-dried, dissolved in nuclease-free water. RNase A (2μL of 10mg/ml stock, Qiagen) digestion was performed at 37oC for 30 min followed by Proteinase K digestion (2 μL, Roche) for 1h at 42oC in the presence 1X Proteinase K digestion buffer (10mM Tris.Cl pH 8.0, 5mM EDTA, 0.5% SDS). DNA was purified using Qiagen PCR purification kit and eluted in 50μL nuclease-free water. DNA concentration was measured using NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific). Equal amount of purified DNA (10ng) was subjected to library construction using the NEBNext®  ChIP-Seq Library Prep Reagent Set for Illumina following the manufacturer's protocol. Adaptor-ligated and index primer-containing amplified DNA corresponding to mononucleosomes was resolved in an 1.5% agarose gel and purified using the Qiagen Gel Extraction Kit, except incubation to melt the gel was performed at 37oC for 30min. The library was then subjected to 50bp single read sequencing in an Illumina Hiseq2000 sequencer.