title: GSM3611242: Replicate of EdU pulse (5min) Thymidine chase (5min) input DNA time course rtt109D mutant [2 min after Thymidine chase]; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Cells were washed in water, cell walls were broken with bead beating and spheroplasts were treated with micrococcal nuclease. EdU from isolated nascent DNA fragments was biotinylated using Click chemistry and nascent DNA was purified with streptavidin coated beads. An input DNA aliquot was set aside prior to biotinylation. nascent and input DNA samples, were treated with calf intestinal phosphatase (NEB), blunt-ended, A-tailed, and ligated (Epicentre) to Illumina genomic PE adaptors with custom 5bp inline barcodes (first 5bp in raw data read), and PCR amplified and size selected by gel purification. For nascent libraries, the nascent strand was separated from its complement with primer extension in the presence of dUTP and the dUTP strand was then digested with the USER enzyme cocktail prior to PCR amplification. All the steps for nascent libraries were done on streptavidine coated magnetic beads.
attributes.tag: GEO Accession
attributes.value: GSM3611242