title: NextSeq 500 sequencing; Nucleosome eviction in mitosis assists condensin loading and chromosome condensation
design: Nucleosome eviction in mitosis assists condensin loading and chromosome condensation
library_construction_protocol: Since condensin accumulates on chromosomes during mitosis, fission yeast cells were collected during prometaphase prior to chromatin digestion by MNase. Cells bearing the cryosensitive mutation nda3-km311 and expressing the cnd2-GFP subunit of condensin were exponentially grown at 32°c and shifted to 19°C for 7 hours to induce a cell-cycle arrest in pro/metaphase by the nda3-km311. Mitotic indexes were measured by scoring the nuclear accumulation of cnd2-GFP. Cells were fixed with 0.5% formaldehyde for 30 minutes, washed with H2O and stored at -80°C. Cells were spheroplasted and MNase digestion of mitotic chromatin was performed as described (Lantermann et al, 2009) using cells arrested in early mitosis by the nda3-km311 mutation at 19°C and crosslinked with 0.5% formaldehyde for 30 min 2x109. Chromatin was digested with increasing amount of MNase to reach a mononucleosome to di-nucleosome ratio of ~80:20. Mononucleosomal DNA fragments were separated on an agarose gel, extracted from the gel and subjected to massive parallel sequencing on an Illumina NextSeq 500 Apparatus.  Ref: Lantermann A, et al. (2009) Genome-wide mapping of nucleosome positions in Schizosaccharomyces pombe. Methods San Diego Calif 48: 218–225 Libraries were prepared according to instructions accompanying the NEB-Next Kit for Illumina. Briefly, gel-purified mononucleosomal DNA fragments (146 bp-long) were end-repaired. Blunt ends were treated with Klenow fragment and dATP to produce protruding 3’ A' for ligating Illumina's adapters. Resulting DNA was PCR amplified 4 cycles. Libraries were sequenced on Illumina NextSeq 500 sequencer following the manufacturer's protocols.
attributes.tag: Experimental Factor: gcn5 deletion
attributes.value: genotype