title: DISTAL-seq of Ecoli
design: Briefly, 50ul reaction consists of: 5ul 10x reaction buffer, 5ul MnCl2, 2.5ul HyperLigase, 15ul 40%(w/v) PEG8000, 5ul adaptor, 1.5ul 5M NaCl, and 15ul DNA solution. Reaction was incubated at 75C for 6 hours and purified by 2 rounds of 1x Ampure beads clean-up with elution volume of 26ul in water. De-blocking of the adaptor 3-ends was done by using 26ul DNA from the previous step, 3ul 10x T4 ligase buffer, and 1ul T4 PNK. Reaction was incubated at 37C for 40min, after which another 1x beads clean-up was done to the reaction mix with elution volume of 10ul. Circularization was done by using 10ul DNA solution from the previous step, 1ul 10x circligase reaction buffer, 0.5ul CircLigase II and 0.5ul MnCl2 (Biosearch Technologies, cat # CL9021). Reaction was incubated at 60C for 1 hour, after which another 1x Ampure clean-up was done with elution volume of 18ul. Dumb-bell DNA digestion is done by using 18ul DNA solution from the previous step, 2 ul of 10x rCutSmart, and 0.2ul of endonuclease. For regular DISTAL-seq, in which internal dU is embedded within the adaptor, USER enzyme was used; for DUET-seq, in which internal dI is embedded within the adaptor, Endonuclease V was used (cat #M0305, NEB). Digestion reaction was incubated at 37C for 15 min followed with heat-inactivation at 65C for 20min. For PCR enrichment. 25ul of Kapa HiFi HotStart ReadyMix (KR0370, Roche Sequencing) and 5ul UDI primer mix (part number 10005922, IDT) were added to the digestion reaction mix. PCR conditions followed manufacturers protocol. For E. coli library, 12 cycles were performed; for DUET-seq, 16 cycles were performed. After PCR, purification was done by 0.9x beads clean-up.