GSM3931521: Q108_U; Equus caballus; RNA-Seq
Total cellular RNA was extracted from embryos using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer's recommendations. After extraction, RNA concentration and quality were analyzed using a NanoDrop DP-1000 spectrophotometer (Agilent Technologies, Palo Alto, CA) and a Bioanalyzer® (Agilent, Santa Clara, CA). All samples had a 260/280 ratio > 2.0, a 28S:18S rRNA ratios >1.8, and RNA integrity number (RIN) > 8 (8.95 ± 0.4, mean ± SEM). A total of 1 μg of RNA was treated with DNase I (Ambion Inc., Austin, TX) for 30 minutes at 37ºC to remove genomic DNA according to manufacturer's instructions. The extracted RNA was kept at -20ºC until further analyses. The mRNA was enriched using oligo(dT) beads. The mRNA was fragmented randomly in fragmentation buffer, followed by first strand cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina, San Diego, CA) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. Double-stranded cDNA was purified using AMPure XP beads (Beckman Coulter, Beverly, CA). In order to select cDNA fragments of 150 base pairs in length, the library fragments were purified with AMPure XP system (Beckman Coulter). The final library was obtained by PCR amplification and purification of PCR products by AMPure XP beads (Beckman Coulter, Beverly, USA).