Illumina HiSeq 2500 sequencing; Genome-wide analysis reveals that budding yeast Rif1 binds to replication origins and at blocked replication forks to protect nascent DNA
Genome-wide analysis reveals that budding yeast Rif1 binds to replication origins and at blocked replication forks to protect nascent DNA
Collected cells were fixed with formaldehyde at room temperature, washed with TBS buffer and resuspended in lysis buffer. A multibead shocker was used to disrupt the cells and chromatin was sheared by sonication. Finally antibodies bound to magnetic Dynabeads were added and incubated.  All cells were cultivated in YPD. Alpha-factor arrested cells were treated for two hours with alpha factor at 30 degrees celsius. HU-arrested cells were first synchronised in alpha factor and then released in to YPD containing 0.2 M hydroxyurea and cultivated for 60 minutes. Normal S Phase cells were arrested by alpha factor at 16 degrees celsius and released into fresh YPD medium at 16 degrees celsius, and then collected at 60 and 90 minutes. Chromatin immunoprecipitation was performed using monoclonal anti-Myc antibody and Dynabeads Protein G. Precipitated DNA was cleaned used TE buffer, glycogen and Proteinase K before extraction with sodium chloride and phenol/chloroform/iso-amylalcohol. DNA was washed using ethanol and purified with a Qiagen PCR purification kit. Library construction using NEBNext Ultra II DNA Library Preparation Kit.
Experimental Factor: genotype
Rif1-13myc
Experimental Factor: growth condition
-factor arrest
Experimental Factor: immunoprecipitate
anti-myc