title: GSM8745879: in vitro cultured D7 blastocyst, manual cumulus denudation, rep5; Bos taurus; RNA-Seq
library_construction_protocol: Total RNA was extracted using a MagMAX mirVana Total Isolation Kit (Applied Biosystems, USA) following the manufacturer's instruction with minor modifications. The RNA samples were quantified by Qubit RNA BR Assay (Thermo Fisher Scientific, Spain) and the RNA integrity was estimated using an RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent, USA), with a minimum RIN value of 8.4. RNA-seq libraries were prepared with a KAPA Stranded mRNA-seq Illumina Platforms Kit (Roche, Switzerland) following the manufacturer's recommendations. Briefly, 50–100 ng of total RNA was used for poly-A fraction enrichment with oligo-dT magnetic beads, after the mRNA fragmentation. The strand specificity was achieved during the second strand synthesis performed in the presence of dUTP instead of dTTP. The blunt-end double-stranded cDNA was 3′-adenylated, and Illumina platform-compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies, Spain) were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with a DNA 7500 assay.