GSM5391101: IVF.BDC.2; Bos taurus; RNA-Seq
RNA was isolated based on size using the RNA Clean & ConcentratorTM 5 kit (Zymo) from three pools each of oocytes, 2-cell stage embryos, 8-cell stage embryos, morula stage embryos, or blastocyst stage embryos according to the manufacturer's protocol for purification of small RNA (<200 nt) and large RNA (>200 nt) as separate fractions.  The large RNA fraction was used to generate sequencing libraries using the Ovation SoLo RNA-Seq System (NuGen Technologies, San Carlos, California) as directed by the manufacturer's protocol for total RNA input. Briefly, first strand cDNA synthesis, cDNA processing, second strand synthesis, and end repair were conducted according to manufacturer's protocols, and samples were then stored at -20°C overnight.  Then, adaptor ligation and purification were performed, followed by PCR amplification of the sequence libraries (optimized to 16 cycles per instructions) and bead purification (twice) of the resulting samples.  Library DNA concentrations were quantified using the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, Massachusetts).  Because DNA concentrations were low, the PCR amplification step was repeated once more with an additional 19 cycles to improve yield.  Next, InDA-C treatment was conducted (Ovation SoLo RNA-Seq System kit), a third round of PCR was performed, and the resulting libraries were bead-purified according to the manufacturer's protocol.