Amplicon sequencing of Cas9 on- and off-target sites in HEK293 cells.
On-target and off-target sites were amplified by PCR with Phusion High Fidelity DNA Polymerase (Thermo Scientific) using primers listed in Supplementary Table 2. The following PCR program was used: 98 C, 10 s; appropriate annealing temperature for each primer pair, 15 s; 72 C, 30 s)  35 cycles, with the addition of DMSO if necessary. The libraries were prepared with 10 ng DNA for each sample using the KAPA HyperPrep-Kit (Roche), according to manufacturers instructions and without fragmentation and size selection. This was followed by 8 cycles of PCR to add sequencing adapters.  After quality control, libraries with similar size were pooled together, resulting in four pools of 14 libraries, respectively one pool of 20 libraries. These pools were quantified with the KAPA Library Quantification Kit (Roche), normalized to 2 nM and pooled again equimolarly to load them on the MiSeq