RNA sequencing of single blastocyst-stage embryos (E3.5) from zygotes which were injected with sperm sRNAs from normal or depressive male mice.

We demonstrated that sperm sRNAs contribute to paternal transmission of depression-like symptoms.To further investigated the mechanism by which sperm sRNAs contribute to depression inheritance. Since the early embryonic period represents a window of plasticity important for adult phenotypes, we injected sperm sRNAs from mice of normal(F0-Ctl) or stress induced depressive(F0-Dep)into zygotes and assessed transcriptional changes when the embryos developed to the E3.5 blastocyst stage (sRNA-Dep-E3.5 vs sRNA-Ctl-E3.5). sRNAs isolated from normal (F0-Ctl) or stress induced depressive (F0-Dep) mouse sperm with a concentration of 2 ng/μl were microinjected into zygotes of the C57Bl/6J background. The zygotes were then cultured in M16 medium at 37 °C in 5% CO2. On the second day, the 2-cell embryos were transferred to potassium-supplemented simplex optimized medium. Embryos at the blastocyst stage (E3.5) were collected at approximately 96 h after microinjection.A single blastocyst-stage embryo was suspended in RNA lysis reagents and frozen at −80 °C, ready for RNA-sequencing processing.