GSM2627465: N5801-dim1-1_MNase; Neurospora crassa; MNase-Seq
For MNase digestion: Frozen nuclei in storage buffer were defrosted and resuspended in MNase digestion buffer (10mM Hepes-NaOH pH 7.5, 10mM KCl, 1.5mM MgCl2, 50ug/mL Bovine Serum Albumin, Filter sterilized; β-merceptoethanol [5mM final], pepstatin [10ug/mL final], leupeptin [10ug/mL final], and PMSF [1mM final] were added prior to use), and a DNA concentration was determined by removing a 10uL fraction, adding SDS to 1% to solubilize nuclear membranes, and quantified with a Qubit HS in triplicate, using the mean concentration. Nuclei containing 16ug of DNA were brought up to 400uL with MNase digestion buffer, CaCl2 was added to a final concentration of 2mM, and MNase (New England Biolabs) was added to a final concentration of 0.5U/uL, and the reaction was digested at 37oC for 20 minutes. At two minute intervals, 1.2ug of DNA (30uL) was removed and added to an eppendorf tube containing 10mM EGTA to stop the reaction, and stored on ice until all timepoints were removed. RNaseA (5ug) was added to all samples and digested an additional 10 minutes at 37oC, and SDS was added to a final concentration of 1% to solubilize the nuclear membrane. DNA was purified from timepoint samples with a Qiagen MinElute Kit, and either used for Southern blotting or sequencing. For MNase-seq, the mono- and di-nucleosome populations, as well as the corresponding linker DNA, of two independent biological replicates of the 8 and 10 minute timepoints were pooled for sequencing, reasoning that at earlier time points, we would capture both the slower-digested euchromatic and rapidly-digested heterochromatic regions. While mono- and di-nucleosome populations were analyzed first (both independently and combined), only mono-nucleosomes were analyzed in all figures for simplicity. Libraries were constructed using the Illumina TruSeq kit, following the manufacturer’s protocols with the following modifications. After End Repair, DNA was purified using a Qiagen MinElute Kit, as recommended by Lombardi et al, 2015. Libraries containing mono- and di-nucleosomes (and the fragments in between these two prominent bands) were size selected with 2% TAE gel electrophoresis following an eight-cycle PCR amplification step; the average size of mono-nucleosomes was ~270bp, while the average size of di-nucleosomes was ~490bp.