3' RNA sequencing of Bos taurus blastocyst
In vitro maturing oocytes were supplemented with follicular fluid collected from preovulatory follicles of greater or lesser maturity. Following identical embryo culture procedures, RNA-sequencing was performed on pools of 2 blastocysts. Briefly, total RNA was isolated from pools of 2 blastocysts with stage score 7 and quality score 1 (IETS) using an optimized TRIzol protocol. Approximately 250 pg total RNA was used as input for reverse transcription, and cDNA amplification was performed using the SMART-Seq v4 3 DE Kit following the manufacturers protocol with volumes adjusted to complete procedures using half reactions. Approximately 500 pg cDNA was utilized for library preparation using the Illumina Nextera XT DNA Sample Preparation Kit and following the manufacturers protocol. Libraries were pooled in equal concentration and sequenced on an Illumina NovaSeq 6000 using an S4 200-cycle flow cell to generate 1x150 (forward, R1) and 1x28 (reverse, R2) paired-end reads at a sequencing depth of approximately 30 million reads per sample.