Schizosaccharomyces pombe 972h-
Protocols: 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.
ENA first public
2016-04-18
ENA last update
2016-04-08
External Id
SAMEA3923255
INSDC center alias
Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210
INSDC center name
Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210
INSDC first public
2016-04-18T17:01:19Z
INSDC last update
2016-04-08T11:13:34Z
INSDC status
public
Submitter Id
E-MTAB-4620:wt replicate #2
broker name
ArrayExpress
genotype
wild type genotype
replicate
replicate 2
sample name
E-MTAB-4620:wt replicate #2