Pooled SMRT-Tag preparation of Homo sapiens: Promega high molecular weight genomic DNA
Genomic DNA is tagmented using a custom-loaded Tn5 transposase with hairpin adapters compatible with PacBio SMRT sequencing in 10mM TAPS-NaOH pH 7.5, 5mM MgCl2, and 10% DMF at 55C for 30 minutes. Fragments are pooled together and cleaned up using a 2X SPRI. Pooled libraires are gap-repaired using a polymerase / ligase combination (Phusion polymerase / Taq ligase) at 37C for 1hr. The pool is then digested with exonuclease III at 37C for 1hr to remove un-repaired, non-productive templates.