GSM7698559: Orc2-Flag ChIP-Exo from cells released from G1-phase (24min), biol rep2; Saccharomyces cerevisiae; ChIP-Seq
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 5' DNA ends were digested using lambda exonuclease. DNA was eluted from resin, de-crosslinked and purified. The second adapter was ligated and DNA purified. After 18 cycles of amplification, DNA libraries were gel extracted using the NEB Monarch PCR & DNA Cleanup Kit according to the manufacturer's instructions to allow elution with smaller volumes. Libraries were quantified using Qubit fluorometry (Thermo Fisher, dsDNA HS Assay) and qPCR (KAPA Illumina library quantification) before pooling and Illumina next generation sequencing.