RNA-Seq of Bos taurus day 3 uninjected control embryos
Day 3 (8-cell/16-cell) embryos were washed twice through PBS+PVA and transferred to a PCR tube with minimal media. The sample was immediately flash frozen in liquid nitrogen and stored at -80 C. Total RNA was isolated from samples using Quick-RNATM MicroPrepkit (Zymo Research), including DNase treatment and 10 l elution volume. Five l of total RNA was used for cDNA preparation using the Ovation RNA-Seq System V2 (NuGEN) according to the manufacturers instructions. cDNA samples were quantified using a Qubit system with dsDNA BR reagents. Two g cDNA in 100 l low TE buffer was used for sonication, using the following parameters: 9 cycles of 30 seconds on high and 90 seconds off, spinning down samples every 3 cycles. After confirming cDNA was sonicated to the right size range for library prep (~74-400bp), 1 g sonicated cDNA was used for library preparation using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs Inc.). Library concentration and quality were determined using the Qubit system with dsDNA HS reagents and the Aligent Bioanalyzer system with HS DNA Assay reagents, respectively. Libraries were pooled, and sequencing was performed using NextSeq SE 75 base pairs (Illumina, USA).