HEK site 4, Cas9-POLD3
The blunt-ended dsODN used in our GUIDE-seq experiments was the same as which was used in the original publication, and then was prepared by annealing the two modified oligonucleotides of the following compositions: 5- P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T -3 and5- P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C -3P represents a 5 phosphorylation and * indicates a phosphorothioate linkage.Genomic DNA was isolated using Qiagen DNeasy Blood & Tissue Kit (250) (#69506) and sheared with a Bioruptor Pico instrument (15 sec ON, 90 sec OFF, 7 cycles) to an average length of around 400-500 bp. The sheared gDNA were then end-repaired, A-tailed and ligated to half-functional adapters, incorporating an 8-nt random molecular index according to the details of the GUIDE-seq protocol provided in the original GUIDE-seq paper.