Polysome-seq in zebrafish early embryonic development

We followed the polysomal association of maternal and early zygotic transcriptome over the first few hours of embryonic development, prior to and after MBT. We isolated polysome-associated (bound) and non-polysome-associated (unbound) mRNAs using sucrose gradient centrifugation followed by size fractionation. Using next generation sequencing (RNA-seq), we profiled the transcriptome in polysome-bound and unbound fractions. Our analysis revealed distinct dynamics of polysome association of cytoplasmically polyadenylated maternal mRNAs. Overall design: rRNA-depleted RNA was extracted from pooled embryos of desired stages (Egg, 1-cell, 16-cells, 128-cells, 3.5hpf and 5.3hpf) and subjected to polysome profiling to separate between polysome-free and polysome-associated fragments. Unfractionated, rRNA-depleted samples at corresponding stages were used as background control. One RNA-seq library was generated for each sample.