title: GSM6156933: N2A Dam Nanopore-DamID; Mus musculus; OTHER
library_construction_protocol: Dam fusion protein expression was induced with 2μg/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. For Nanopore-DamID 2.5μg of gDNA was dephosphorylated by the addition of 5μl of 10X NEB Cutsmart® buffer and 1μl of QuickCIP (NEB) in a total volume of 48μL. The reaction was mixed by gentle flicking and incubated at 37°C for 30 minutes. The QuickCIP was then heat inactivated at 80°C for 5 min. 2μl of DpnI (NEB) was added to the reaction, mixed by gently flicking and incubated at 37°C overnight. DNA was A-tailed by addition of 1μL 10mM dATPs and 1μL of Klenow exo- (NEB) and incubation at 37°C for 1hr. The digested DNA was purified using the Qiagen QiaQuick™ PCR Purification kit (which purifies fragments <10kb) and eluted in 60μL 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. For Illumina-DamID libraries were prepared according to the protocol in Marshall et al., 2016, Nature Protocols) using the NEBnext UltraII kit for Illumina library construction. Nanopore DamID or Illumina-DamID