Whole Genome Amplification from Single Blastocyst

The fixed single blastocyst was transferred to a PBS drop (1 μL) in a 0.5 mL PCR tube (#PCR-05-C.; AxyGen Scientific, Inc., Union City, CA, USA) with the help of a mouth piece-controlled micropipette, as previously described [33 (link),34 (link)]. Genomic DNA was extracted by adding 100 μL of lysis buffer mentioned above and lysed at 37 °C for over two days. The solution was extracted with phenol/chloroform [43 (link)] and the supernatant was precipitated with isopropanol. The precipitated DNA was dissolved in 20 μL of sterile water and stored at 4 °C. To increase the amount of whole genomic DNA, we employed WGA using illustra GenomiPhi V2 DNA Amplification Kit (#25-6600-31; GE Health Care Japan, Tokyo, Japan), as previously described [34 (link),43 (link)]. Briefly, 2 μL of genomic DNA was mixed with 8 μL reaction buffer containing enzyme in a 20 μL volume and allowed to react overnight at 30 °C. The resulting WGA products (2 μL) were subjected to the first PCR as described above.