Bigdye terminator v3.1 cs kit

For genomic DNA extraction, 0.5–1 × 10⁶ cells were washed with 1X DPBS, DNA was purified using the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69506) as recommended by manufacturer′s instructions, and purified DNA was resuspended in 100 µl of water. Target sequences were amplified using KAPA HiFi HS ReadyMix (Kapa Biosystems, Cat. No. KK2602) polymerase chain reaction (PCR), enzymatic PCR product cleanup was performed with ExoSAP-IT Express reagent (Thermo Fischer Scientific, Cat. No. 75001), and Sanger sequencing was performed using the BigDye Terminator v3.1 CS Kit (Thermo Fischer Scientific, Cat. No. 4337456) according to the manufacturer′s instructions. Genotyping primers are listed in Supplementary Table 3. Reactions were then purified by ethanol precipitation and acquired on a 3130xl Genetic Analyzer (Applied Biosystems). Sequence alignments were analyzed with Snapgene (GSL Biotech LLC), and sequence trace files with low base calling confidence were excluded from analyses.

For genomic DNA extraction, target sequences were PCR-amplified with KAPA HiFi HS ReadyMix (Kapa Biosystems, Cat. No. KK2602), amplicons were treated with ExoSAP-IT Express reagent (Thermo Fischer Scientific, Cat. No. 75001) for enzymatic cleanup, and Sanger sequencing was prepared with the BigDye Terminator v3.1 CS Kit (Thermo Fischer Scientific, Cat. No. 4337456). Reactions were then purified by ethanol precipitation and acquired on a 3130xl Genetic Analyzer (Applied Biosystems). Sequence alignments were analyzed with Snapgene (GSL Biotech LLC), and sequence trace files with low base calling confidence were excluded from analyses. Genotyping primers are listed in Supplementary Table 9.