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===Fragmentation and end-polishing (Make blunt ends with 5'P)=== *The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp *End-Repair Reactions {| {{table}} | Fragmented DNA|| 85ul |- | 10X End Repair Bufer|| 10ul |- | End Repair Enzyme Mix|| 5ul |- | |} *Incubate tubes at RT for 30minutes. *Perform a Qiaquick purification and elute with 39ul EB buffer. *NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads. *A-Tailing Reactions {| {{table}} | Blunet-end DNA||37ul |- | 10X dA-Tailing Reaction Buffer||5ul |- | Klenow Fragment (3'-5' exo-)||3ul |- | H2O||5ul |- | |} Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 40 ul EB. *Measure concentration with nanodrop
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