GSM8244657: TE A6; Bos taurus; RNA-Seq
Total RNA was extracted using MagMAX mirVana Total Isolation Kit (Applied Biosystems) following the manufacturer's instruction with minor modifications. Briefly 200 µl of Lysis Binding Mix were added to the sample, followed by gentle pipetting and 5 min incubation at room temperature. Then 20 µl of Binding Beads Mix were added and shaken gently for 5 min. Beads‐mRNA complexes were washed once in each Wash Solution 1 and 2. Following the washing step, samples were treated with 50 µl of Turbo DNAse treatment, 50 µl of Rebinding Buffer and 100 µl of Isopropanol were added to the sample and mixed gently. Finally, following a double wash in Wash Solution 2, total RNA was eluted in 20 µl of Elution Buffer and stored at −80°C until analysis. RNA‐seq libraries were prepared with KAPA Stranded mRNA‐Seq Illumina Platforms Kit (Roche) following the manufacturer's recommendations. Briefly, 50–100 ng of total RNA was used for the poly‐A fraction enrichment with oligo‐dT magnetic beads, following 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) were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on a HiSeq. 4000 system (Illumina) with a read length of 2 × 76 bp + 8 bp + 8 bp using the HiSeq. 4000 SBS kit (Illumina) obtaining >30 M reads/sample.