GSM8928139: Top2 cdc20+noco IP; Saccharomyces cerevisiae; ChIP-Seq
Cells were lysed by beating in MP biomedicals FastPrep-24 machine for 30 seconds 3 times with 10 minute intervals on ice. Cell pellets were washed once in FA lysis buffer/1x PI/1mM PMSF/0.1% SDS before sonicating in a Bioruptor (Diagenode) for 2 times 20 cycles at 30sec on/off intervals at "High" setting. The supernatant was collected, a INPUT fraction was taken and the rest was subjected to IP with mouse monoclonal SV5-Pk1 antibody conjugated to Dynabeads over night. Dynabeads were washed and bound protein eluted using TES buffer. Samples were decrosslinked with Proteinase K and purified using Promega Wizard kit. DNA was blunted using NEB Quick Blunting kit followed by Ampure XP bead purification of fragments over 100bp. dA tails were added using Klenow (exo-) and NextFlex adaptors ligated to each sample. This was followed by two Ampure purifications of fragments over 100bp and then fragments over 150-200bp. Libraries were amplified using NextFlex primers and Phusion DNA polymerase. Lastly, double-sided Ampure purification was carried out to obtain fragments between 100-300bp in size.