Genomic DNA (gDNA) was extracted using the Mag-Bind® Blood & Tissue DNA HDQ 96 Kit (Omega Bio-tek) and quantified by the Qubit™ dsDNA Quantification Assay Kits (ThermoFisher). Illumina-compatible guide array amplicons were generated by amplification of the gDNA in a one-step PCR. Indexed PCR primers were synthesized by Integrated DNA Technologies using the standard 8nt indexes from Illumina (D501-D508 and D701-D712). The sequences for the primer sets were listed in Supplementary Data 10 .
At least ~200X coverage gDNA per replicate across multiple reactions were amplified. Each gDNA sample was first divided into multiple 100 μL reactions with most 10 μg gDNA per reaction. Each reaction contained 0.5 μL forward primer (100 μM), 10uL reverse primer (5 uM) 8 μL dNTPs, 5 μL DMSO, 10 μL 10X Titanium Taq Buffer, and 1.5 μL Titanium Taq DNA Polymerase (Takara). The PCR conditions were: An initial denaturation at 95 °C for 60 s, followed by 28 cycles of 30 s at 94 °C, 30 s at 52 °C, and 30 s at 72 °C followed by a final extension at 72 °C for 10 min. After the PCR, all reactions from the same sample were pooled and purified with Agencourt AMPure XP SPRI beads according to the manufacturer protocol (Beckman Coulter). Purified amplicons were quantified by Qubit™ dsDNA Quantification Assay Kits (ThermoFisher) and sequenced on a HiSeq2500 with a Rapid Run (200 cycle) kit (Illumina).
At least ~200X coverage gDNA per replicate across multiple reactions were amplified. Each gDNA sample was first divided into multiple 100 μL reactions with most 10 μg gDNA per reaction. Each reaction contained 0.5 μL forward primer (100 μM), 10uL reverse primer (5 uM) 8 μL dNTPs, 5 μL DMSO, 10 μL 10X Titanium Taq Buffer, and 1.5 μL Titanium Taq DNA Polymerase (Takara). The PCR conditions were: An initial denaturation at 95 °C for 60 s, followed by 28 cycles of 30 s at 94 °C, 30 s at 52 °C, and 30 s at 72 °C followed by a final extension at 72 °C for 10 min. After the PCR, all reactions from the same sample were pooled and purified with Agencourt AMPure XP SPRI beads according to the manufacturer protocol (Beckman Coulter). Purified amplicons were quantified by Qubit™ dsDNA Quantification Assay Kits (ThermoFisher) and sequenced on a HiSeq2500 with a Rapid Run (200 cycle) kit (Illumina).