Mnase-seq of SWP73 subunits of chromatin remodeling complex in Arabidopsis
Mapping nucleosome positioning was performed with 2g samples of 14-day-old WT, swp73a-1, and swp73b-1 seedlings according to Saleh et al. (2008) and Zhu et al. (2013) with minor modifications (i.e. the buffer contained 1% proteinase inhibitor cocktail (Sigma) instead of pepstatin A and aprotinin). 200 Kunitz Units of Micrococcal Nuclease (NEB) were used for digestion of each sample. DNA was purified with phenol:chloroform extraction, ethanol precipitation and size separated on 2% agarose gels. Bands corresponding to ~150 bp were gel isolated, purified and used for qPCR. Illumina compatible libraries of MNase-treated gDNA were prepared according to the protocol of Ovation Ultralow DNA Library kit (NuGEN). Libraries were quantified by fluorometry, immobilized and processed onto a flow cell with a cBot (Illumina) followed by sequencing-by-synthesis with TruSeq v3 chemistry on a HiSeq2500.