Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

Laboratory of Biochemistry and Molecular Biology of Germ Cells Institute of Animal Physiology and Genetics CAS Rumburská 89 277 21 Liběchov Czech Republic

Laboratory of Biochemistry and Molecular Biology of Germ Cells Institute of Animal Physiology and Genetics CAS Rumburská 89 277 21 Liběchov Czech Republic Laboratory of Early Mammalian Developmental Biology Department of Molecular Biology and Genetics Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Laboratory of Early Mammalian Developmental Biology Department of Molecular Biology and Genetics Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Laboratory of RNA Biochemistry Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 128 44 2 Prague Czech Republic

Laboratory of RNA Biochemistry Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 128 44 2 Prague Czech Republic Laboratory of Biochemistry and Molecular Biology of Germ Cells Institute of Animal Physiology and Genetics CAS Rumburská 89 277 21 Liběchov Czech Republic

Queen's Medical Research Institute MRC Centre for Reproductive Health The University of Edinburgh 47 Little France Crescent Edinburgh EH16 4TJ UK

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