Nanopore-DamID of CTCF-DamN126A in HEK293T cells replicate1
Dam fusion protein expression was induced with 2g/mL doxycycline and genomic DNA (gDNA) was extracted from one million cells cultured in Nunc T75 flasks using an NEB Monarch gDNA extraction kit according to manufacturer's instructions, with the exception of using inversion rather than vortexing for mixing. 2.5g of gDNA was dephosphorylated by the addition of 5l of 10X NEB Cutsmart buffer and 1l of QuickCIP (NEB) in a total volume of 48L. The reaction was mixed by gentle flicking and incubated at 37C for 30 minutes. The QuickCIP was then heat inactivated at 80C for 5 min. 2l of DpnI (NEB) was added to the reaction, mixed by gently flicking and incubated at 37C overnight. DNA was A-tailed by addition of 1L 10mM dATPs and 1L of Klenow exo- (NEB) and incubation at 37C for 1hr. The digested DNA was purified using the Qiagen QiaQuick PCR Purification kit (which purifies fragments <10kb) and eluted in 60L of H2O. The DNA was prepared for ONT sequencing with the DNA Ligation Sequencing Kit (LSK110), starting from the adaptor ligation step and using the Short Fragment Buffer.