HIDE-Seq: Bxb1 alone rep 1
HIDE-seq reactions with 8 ug of purified gDNA from PBMCs (Qiagen Blood and Tissue kit) or, in separate reactions, 8 ug of purified gDNA HEK293attB cells was incubated with 10 nM attB_66bp annealed oligonucleotide (for cryptic attP site or FDE discovery) or 10 nM attP_72bp annealed oligonucleotide (for cryptic attB site or FDE discovery) and 400 nM of recombinant Bxb1 integrase (GenScript) in recombination buffer7 (10 nM Tris-HCl, pH 8.0, 100 mM KCl, 5% glycerol) for 8 hours at 37C and then held at 4C. To anneal the attB_66 and attP_72 oligonucleotide substrates for HIDE-seq, 25 ul of attB_66bp_P_Top (100 mM) and 25 ul of attB_66bp_P_Bot (100 mM) or 25 ul of attP_72bp_P_Top (100 mM) and 25 ul of attP_72bp_P_Bot (100 mM), respectively were mixed together in wells of a 20 ul 96 well plate. Note, oligonucleotides were resuspended in duplex buffer (Integrated DNA technologies (IDT)). The plate with oligonucleotide samples were placed into a thermocycler, and the following anneal program was performed with a 105C heated lid: 95C/2 minutes, 750 cycles at -0.1C per cycle/1s, 4C hold. Reactions were inactivated with RNAase A (New England Biolabs (NEB) Monarch) and then Proteinase K (NEB) and gDNA was purified with 0.7x AMPure (Beckmann Coulter) bead cleanup. Control Digenome-seq reactions were performed with gDNA and spCas9 (IDT) with VEGFA S2 sgRNA (IDT) were performed according to the published protocol21. Isolated gDNA from both HIDE-seq and Digenome-seq reactions was quantified by Qubit fluorometry (Thermo Fisher Scientific) and submitted for PCR-free WGS at 30x coverage (Azenta).