Cell tissue blood whole genome dna extraction kit

The experiment randomly selected eight embryos labelled a to h to extract the whole genome of single cell clones using the cell tissue blood whole genome DNA extraction kit (Tiangen, Beijing, China). The whole genome was used as the template and btHP-1963-F/btHP-1963-R shown in Table S3 was used as primer for polymerase chain reaction (PCR) amplification. The 25 μL PCR reaction system contained 12.5 μL of 2 × Phanta Max Master Mix (Vazyme, Nanjing, China), 1 μL of target fragment, 1 μL each of 100 mmol/L upstream and downstream primers and 9.5 μL of distilled water (ddH₂O). The PCR reaction conditions included pre-denaturation at 93 °C for 30 s, denaturation at 93 °C for 20 s, annealing at 58 °C for 30 s and extension at 72 °C for 70 s, for 33 cycles; followed by an extension at 72 °C for 2 min and storage at 4 °C. The PCR products were identified by agarose gel electrophoresis (AGE) at a concentration of 1.2% and the amplified products were sequenced by Sanger at Huada Genetics (Huada, Shanghai, China).

The whole genome of the skin tissue was extracted separately using the cell tissue blood whole genome DNA extraction kit (Tiangen, Beijing, China) and was used as a template and NEW-F/NEW-R as primers as shown in Table S3 for PCR amplification. The PCR reaction system and reaction conditions were the same with embryo genome analysis. The PCR products were identified by AGE at a concentration of 1.2% and the amplified products were sequenced by Sanger at Huada Genetics (Huada, Shanghai, China).