GSM5987959: MP - 70k; Bos taurus; ncRNA-Seq
GSM5987959
Total RNA was isolated using Trizol LS and treated with DNAse on column with RNeasy mini-kit and treated on-column with DNase, according to the manufacturer's protocol (Qiagen, Hilden, Germany) Library preparations were performed following modified SOPs at ArrayStar platform to allow for larger RNA size window (8–35 nt). The purity, quality, and concentration of total RNA samples were determined with NanoDrop ND-1000 (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent, Santa Clara, CA). MiRNA sequencing library was prepared from total RNA through: (1) 3′-adapter ligation, (2) 5′-adapter ligation, (3) cDNA synthesis, (4) PCR amplification, and (5) size selection of approximately 120 to 155 bp of PCR-amplified fragments (corresponding to approximately 8 to 35 nt of small RNA). The complete libraries were quantified by Agilent 2100 Bioanalyser. nc-RNAseq, The cDNA library samples were then diluted to a final concentration of 8 pM and denatured as single-stranded DNA. Cluster generation was performed on the Illumina cBot using TruSeq Rapid SR cluster kit (#GD-402–4001, Illumina, San Diego, CA) and sequenced for 51 cycles on Illumina HiSeq 2000, using TruSeq Rapid SBS Kits (#FC-402–4002, Illumina), following the manufacturer's instructions.