Small RNA-Seq of Bos taurus blastocyst embryos
In-vitro fertilized blastocyst stage embryos were washed twice through PBS+PVA and transferred to a PCR tube with minimal media. Total RNA was isolated using Quick-RNA MicroPrepkit (Zymo Research), including DNase treatment. All seven l of total RNA was then used for library preparation using the SMARTer smRNA-Seq Kit for Illumina (Clontech Laboratories, Inc.) according to the manufacturer's instructions. The SMARTer smRNA-Seq Kit for Illumina is designed for small RNA input (1 ng) and uses polyadenylation of RNAs for an oligo(dT) primer, followed by a template switching technology in order to have ligation-free adapter and index incorporation to avoid biases associated with adapter ligation. After library preparation, the NucleoSpin Gel and PCR Clean-Up kit (Macherey-Nagel) was used for library purification according to the manufacturers protocol. cDNA 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. Size selection for lengths between 148 bp185 bp (adapter size of 153 bp) of each library was performed with the BluePippin System (Sage Science, USA) using 3% Agarose Gel Cassettes (Sage Science). Quantification of size-selected libraries was again performed using the Qubit system with dsDNA HS reagents and the Aligent Bioanalyzer system with HS DNA Assay reagents. Libraries were pooled and Illumina sequencing performed using NextSeq SE 75 base pairs (Illumina, USA). Two rounds of sequencing were performed, run 1 (_R1) and run 2 (_R2).