amplicon sequencing of URA3 and ADE2_biologicalreplicate1
genomic DNA of total of 14 populations (Supplemental Dataset. S4) were extracted from cells using the Yeast Genomic DNA Extraction Kit (Solarbio life sciences, Beijing, China) according to the manufacturers directions. The CRISPR/Cas9 and CRISPR/Cpf1 targeted genomic region of URA3 and ADE2 loci were PCR amplified for subsequent Sanger sequencing using 20-bp specific primers locating at 100-bp upstream and downstream of the predicted cleavage site and flanking with 6-bp specific barcode sequence (Supplemental Dataset. S3), and then generated 200-bp fragments, regardless of the length of barcode and index sequence. The products of PCR were purified and recovered using Gel Extraction Kit (cwbio, Beijing, China) according to the manufacturers directions. The purified samples were quantified using NanoDrop 2000 (Thermo Scientific, USA) and diluted uniformly to 0.1 g/l, and then 2 l for each sample was assembled in a library pool and mix well. The mixed library was sequenced on the Illumina NovaSeq 6000 (Novogene, Tianjin, China) according to the manufacturers protocols.