Quantitect purification kit

We isolated total RNA from 100 denuded GV-stage oocytes using an Arcturus PicoPure RNA Isolation Kit (Applied Biosystems), and cDNA was generated using a QuantiTect Purification Kit (Qiagen, Düsseldorf, Germany). For Rac1 plasmid construction, PCR products were purified and cloned into the linearized pCS2⁺ vector with six Myc tags using the Uniclone one-step seamless cloning kit (Genesand Biotech Co.,Ltd, Cat# SC612). The plasmid was propagated using Trans1-T1 (Transgen Biotech, Beijing, China) as the host strain and purified with a TIANpure Midi Plasmid Kit (DP107, TIANGEN Biotech, Beijing), followed by sequencing to confirm the open reading frame. For the synthesis of cRNA, the Rac1 plasmids were linearized with XbaI. Capped cRNAs were achieved using in vitro transcription with SP6 mMESSAGE mMACHINE (Ambion, CA, USA) according to the manufacturer’s instructions. Synthesized RNA was aliquoted and stored at − 80 °C (the related primer sequences can be found in Supporting Information Table S1).

Total RNA was extracted from 50 denuded oocytes at the stages indicated above using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, CA, United States), and cDNA was generated with the Quantitect Purification Kit (Qiagen, Düsseldorf, Germany) (the primers we used to amplify the coding DNA sequence [CDS] of Plk1 and mutants are listed in Table S1 [Supporting Information]). PCR products were purified, digested with Fse I and Asc I (New England Biolabs, Beverly, MA, USA), and then inserted into the pCS²⁺ vector with Myc-tags. For the synthesis of cRNA Myc-Pak2 mRNA, plasmid constructs were linearized using Not1 enzyme (NEB) and purified. Capped cRNAs were constructed using in vitro transcription with SP6 mMESSAGE mMACHINE (Ambion, CA, USA) according to the manufacturer’s instructions. Synthesized RNA was ultimately aliquoted and stored at −80 °C.

Total RNA was extracted from 50 denuded oocytes using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, CA, United States), and cDNA was generated by Quantitect Purification Kit (Qiagen, Düsseldorf, Germany). PCR products were purified, digested with FseI and AscI (NEB Inc., MA, United States), and cloned into the pCS2⁺ vector with Myc-tags. For the synthesis of Myc-ASB7 mRNA, ASB7-pCS2 + plasmids were linearized by NotI, and capped cRNAs were in vitro transcribed with SP6 mMessage mMachine (Ambion, CA, United States) and purified by RNeasy Micro Kit (Qiagen, Germany). Synthesized RNA was aliquoted and stored at −80°C. The related primer sequences can be found in Supplementary Table 1.