Gene prep star pi 80x

DNA was extracted from frozen feces using an automated nucleic acid extractor (Kurabo Industries Ltd., Osaka, Japan), with some modifications to a previously reported protocol [27 (link)]. Frozen stool samples, the size of a grain of rice, were homogenized with 500 µL of lysis buffer (No. 10, Kurabo Industries Ltd., Osaka, Japan) and 0.5 g of 0.1 mm glass beads in a 2 mL vial. The mixture was mechanically disrupted at 4,260 rpm for 50 s at room temperature (20–25 °C), using a tabletop cell disruptor, Cell Destroyer PS 1000 (Bio Medical Science, Tokyo, Japan). The cells were then centrifuged at 12,000× g for 5 min at room temperature (20–25 °C). The supernatant (200 µL) was collected and mixed with 150 µL of lysis buffer and 150 µL of proteinase K buffer containing 0.4 mg/mL of proteinase K (No. 2, Kurabo Industries Ltd., Osaka, Japan). DNA was extracted using an automated nucleic acid extractor Gene Prep Star PI-80X (Kurabo Industries Ltd., Osaka, Japan), and DNA concentration was measured using a NanoDrop spectrophotometer ND-1000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The extracted DNA samples were stored at −30 °C until use.

The automated DNA extraction system was used to extract genomic DNA from tail biopsies or whole blastocysts (GENE PREP STAR PI-80X, KURABO, Osaka, Japan). The PCR products amplified with specific primer sets were directly sequenced. Primers for genotyping analysis are shown in the Supplementary Table 2. For genotyping of mouse Rosa26 and rat Thy1 loci, nested PCR was performed using the external primers as follows: 5′-GCTCTCGGGGCCCAGAAAAC-3′ and 5′-GACTTCTAAGATCAGGAAAG-3′ (mouse Rosa26); 5′-ATGACATTCGCTGTCATAAC-3′ and 5′-CAGCAGAGAGAACACATATC-3′ (rat Thy1).