GSM1201972: MNase_spt6_100Uml_N1; Schizosaccharomyces pombe; MNase-Seq
Cells were spheroplasted in 2 ml of CES buffer (50mM Citric acid/50mM Na2HPO pH 5.6; 40mM EDTA pH=8.0; 1.2M Sorbitol) containing 1 mg/ml Zymolyase 100T (United States Biological) and 20 mM  _-mercaptoethanol for 1 hour at 30¡ C.  Spheroplasts were pelleted, washed with 50 ml of Tris/Sorbitol buffer (40 mM TrisHCl pH=8.0; 1.2 M Sorbitol; 50 mM NaCl; 1 mM EDTA) and subjected to MNase digestion in 2 ml of NP-buffer (40 mM TrisHCl pH=8.0; 1.2 M Sorbitol; 50 mM NaCl; 1 mM EDTA, 0.1% NP-40, 0.75% Sigma Fungal Protease Inhibitor Cocktail; 10 mM _-mercaptoethanol; 5mM spermidine; 10 mM CaCl2) containing 150 u/ml of Nuclease S7 (Roche) for 5 min at 37¡ C.  Reactions were stopped by addition of EDTA and EGTA mix to final concentration 25 mM each.  Crosslinking was reversed overnight at 65¡ C in the presence of 0.5% SDS and 1 mg/ml proteinase K.  DNA was purified by phenol/chloroform extraction and ethanol precipitation. RNA was removed by treatment with RNAseA/T1 mix (Fermentas). Purified DNA was resolved on 1% agarose gel and a band corresponding to mono-nucleosomal fragments was excised and subjected to gel-extraction. DNA was end repaired with T4 DNA Polymerase (Invitrogen), T4 PNK (NEB) and DNA Pol I, Large Klenow fragment (Invitrogen), and an 'A' base was added using Klenow 3' to 5' exo minus (NEB).  1 pmol of barcoded adaptors were ligated on with T4 DNA ligase (Roche) and the products were PCR amplified with Phusion DNA Polymerase (NEB) and size selected by purification on 2% agarose-EX E-gels (Invitrogen) for fragments between 200-600 bp.  Library size and concentration was determined by Bioanalyzer or Tapestation analysis (Agilent) and libraries were pooled equimolar amounts with up to 26 in each sequencing lane.  Pooled libraries were gel purified twice, followed by column purification on MinElute columns (Qiagen).  At least 10 nM were submitted for analysis on the Illumina Hi-Seq at Tufts University Core Facility.