Genome-wide analysis of replication initiation in Tetrahymena

We mapped Tetrahymena macronuclear replication origins on a genome-wide scale using Okazaki fragments sequencing (OK-seq). OK-seq permits a high-resolution, quantitative analysis of replication fork initiation on a genome-wide scale that is dependent on the depth of coverage within a population of molecules. Replication initiation sites of the TXR1 (Histone H3 K27 monomethyltranferase) deletion strain were also studied compare to wild type using OK-seq. Overall design: 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).