Identifying the translatome of mouse NEBD – stage oocytes via SSP-profiling; a novel polysome fractionation method

Meiotic maturation of oocyte relies on pre-synthesized maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol utilising small SW55Ti ultracentrifugation tubes with a volume of 5 ml. This newly optimized 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 stage that has thus far remained unexplored. Our analyses have identified 1778 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteoasomal 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 have 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. 4 replicates of RNAseq datasets from polysomal (P) and non-polysomal (NP) fractions