Single-molecule mapping of replisome progression

Replicon-seq is a method to study the progression of sister replisomes during DNA replication. This method relies excision of the full-length of replicons by the fusion of MNase to MCM4 and sequencing via Nanopore technology. Overall design: MCM4 was fused to Miccrococcal nuclease ( MNase) to generate DNA double strand break at the site of replisomes. DNA ends are repaired and MinION compatible DNA adaptors are ligated. Full length molecules are sequenced. Because cells have been released from a G1 arrest in presence of BrdU, we can select for replicon reads (reads that contain BrdU) informatically using DNAscent.