Microinjection and Maturation of Oocytes

Histone 2B (H2B)-RFP, UtrCH-mCherry (a gift from William Bement^{25 (link)}; Addgene plasmid #26740) and Sirt2-H187Y-GFP (a gift from Eic Verdin^{17 (link)}) cRNAs were used. All plasmids were sequenced with T3 primer (Supplementary Table 1) before transcription. The mMESSAGE mMACHINE High Yield Capped RNA Transcription kit (Ambion) was used to produce cRNA constructs by T3-promoter driven in vitro transcription from linearised DNA template^{7 (link),51 (link),52 (link)}. Following in vitro transcription, cRNA size was verified on agarose gels and concentrations were determined using a spectrophotometer. Constructs were microinjected at the following concentrations: H2B-RFP at 250 ng μl⁻¹, UtrCH-mCherry at 700 ng μl⁻¹ and Sirt2-H187Y at 1000 ng μl⁻¹. Following microinjection at the GV-stage, oocytes were maintained arrested in IBMX-treated αMEM HEPES-buffered medium for at least 2 h to allow time for protein translation before washing free from IBMX to allow maturation to occur.