GSM3839836: Oxy_Mut_MNase-seq_invivo_LightDigest_replAB; Oxytricha trifallax; MNase-Seq
Oxytricha in vivo MNase-seq protocol: Vegetative Oxytricha cells were fixed in 1% (w/v) formaldehyde for 10 min at room temperature with gentle shaking, and then quenched with 125 mM glycine. Macronuclei were subsequently isolated by sucrose gradient centrifugation from fixed cells as previously described25. Purified nuclei were pelleted by centrifugation at 4000 x g, washed in 50 ml TMS buffer (10 mM Tris pH 7.5, 10 mM MgCl2, 3 mM CaCl2, 0.25 M sucrose), resuspended in a final volume of 300 μl, and equilibriated at 37oC for 5 min. Chromatin was then digested with MNase (New England BioLabs) at a final concentration of 15.7 Kunitz Units / μl at 37oC for 1 min 15 sec, 3 min, 5 min, 7 min 30sec, 10 min 30 sec, and 15 min respectively. Reactions were stopped by adding 1/2 volume of PK buffer (300 mM NaCl, 30 mM Tris pH 8, 75 mM EDTA pH 8, 1.5% (w/v) SDS, 0.5 mg/ml Proteinase K). Each sample was incubated at 65oC overnight to reverse crosslinks and deproteinate samples. Subsequently, nucleosomal DNA was purified through phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation.  Each sample was loaded on a 2% agarose-TAE gel to check the extent of MNase digestion. The sample exhibiting ~80% mononucleosomal species was selected for MNase-seq analysis. Mononucleosome-sized DNA was gel-purified using a QIAquick gel extraction kit (QIAGEN) for subsequent paired-end sequencing on an Illumina HiSeq 2500 according to manufacturer's instructions. MNase-seq (Paired-end sequencing); standard TruSeq Illumina library construction protocol
GEO Accession
GSM3839836