NGS sequencing of genomic DNA samples

Genomic sites of interest were amplified from genomic DNA samples. Briefly, primers were designed to generate between 228 and 313 bp amplicons using Phusion High-Fidelity DNA polymerase (Thermo Scientific). The primers used are presented in Supplementary Fig. ⁹.

The amplified DNAs have been extracted on an agarose gel and purified (using the PCR clean-up gel extraction kit #740609.50, Macherey-Nagel).

Raw data of the NGS sequencing organization is listed in Supplementary Fig. ¹⁰.

Illumina adapters and barcodes were added to amplicon pools by ligation and sequenced on an Illumina NextSeq (NovaSeq PE250, Novogene company). Alignment of amplicon sequences to a reference sequence was performed using a custom python pipeline that was used to count nucleotide substitutions in the base editor window (both expected C:G to T:A conversions and other substitutions) and indels overlapping the spacer sequence.