title: GSM4085489: Wild-type MNase seq 6h replicate 2; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Approximately 90 OD600 units of cells were crosslinked for 20 min with formaldehyde (1% v/v) and the reaction was quenched with glycine (125 mM). Subsequently, cells were resuspended in 20 ml of buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) plus β-mercaptoethanol (10mM) and treated with 50ul of 5mg/ml T100 Zymolase (MP Biomedicals) for 60 min. Next, cells were resuspended in 2.5 ml NP buffer (0.5 mM spermidine, 1 mM β-mercaptoethanol (β -ME), 0.075% (w/v) Tergitol solution-type NP-40 detergent (NP-40), 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2 , 1 mM CaCl2). 100ul of chromatin (equivalent to 10% of a 90 ODunit prep) was treated with various concentrations of MNase (NEB), equivalent to 1/5, 1/12 or 1/25 µl of a 2 mg/ml stock, for 30 min at 37 °C. The reaction was quenched with EDTA (10 mM).  To check for the extent of MNase digestion, 100 μl of MNase treated extracts were reverse crosslinked overnight in SDS-TE (1% (w/v) SDS, 10 mM Tris pH 8, 1 mM EDTA) and treated with RNase A.  DNA fragments were purified using phenol-chloroform-isoamyl alcohol, were extracted using ethanol precipitation, were resuspended in TE buffer and were separated by gel electrophoresis. The extracts which showed a mostly mono-nucleosome pattern were chosen. The gel fragments containing the mono-nucleosome band (100-200 bp) was excised, and the DNA purified using a gel purification kit (Macherey-Nagel). At least 1 ng of purified DNA fragments was used as the inputs for the KAPA Hyper Prep kit (KK8504, Roche). Libraries were prepared according to manufacturer's instructions. Amplified libraries were cleaned and purified by gel extraction and sequenced on an Illumina HiSeq 2500 to an equivalent of 50 bases paired-end reads, at a depth of approximately 8 million reads per library.