GSM5269304: PIP-seq of Mcm4-Flag, 30min released,treated_rep2; Saccharomyces cerevisiae; OTHER
Lysates were prepared using standard procedures, sonicated chromatin clarified and Flag-tagged proteins immunprecipitated. Immunoprecipitated DNA was treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation. After phosphorylation, the first adapter was ligated using T4 DNA Ligase and T4 Polynucleotidekinase. Gaps were filled with phi29 polymerase and samples subjected to 10mM potassium permanganate. DNA was eluted from resin, de-crosslinked and purified. After cleanup and piperidine cleavage, samples were purified again and second adapter were ligated and DNA purified. After 18 cycles of amplification, were cleaned up using AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions. Libraries were quantified using Qubit fluorometry (Thermo Fisher, dsDNA HS Assay) and qPCR (KAPA Illumina library quantification) before pooling and Illumina next generation sequencing.