RNA-Seq of two-cell biparental mouse embryos
In vivo fertilized two-cell embryos were washed three times in PBS+PVA and transferred to a PCR tube that was then frozen in liquid nitrogen. Samples were stored at 80C until processed for RNAseq. Total RNA, isolated with an ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific, catalog #KIT0204) according to the manufacturers instructions, was used for library preparation using the Ovation SoLo RNA-Seq Systems (NuGEN catalog #0501) according to the manufacturers instructions. cDNA libraries were quantified and evaluated using a 2100 Bioanalyzer (Aligent Technologies). Libraries were pooled, and sequencing was performed using NextSeq single end 75 base pairs.