Genome-wide analysis of replication initiation in a TXR1 (Histone H3 K27 monomethyltranferase) overexpression strain in Tetrahymena

We mapped Tetrahymena macronuclear replication origins in TXR1 overexpression strain on a genome-wide scale using Okazaki fragments sequencing (OK-seq). Replication initiation sites of TXR1 overexpression strain followig cadmium induction of MMT1 promoter driven-TXR1 were studied compare to the uninduced TXR1 overexpression strain without cadmium using OK-seq. Asynchronous exponentially growing Tetrahymena cells were labelled by EdU (5-ethynyl-2’-deoxyuridine, ThermoFisher) for 5 min to label newly synthesized DNA. Cells were washed and collected. Genomic DNA was extracted. Okazaki fragment were isolated and biotinylated using ClickIt chemstry. Sequencing adaptor were ligated and Okazaki fragment capture using streptavidin magnetic beads (Petryk et al. 2016).