GSM7058368: Peripheral white blood cells, Subfertile, 32; Bos taurus; miRNA-Seq
Total RNA was isolated from 14 heifers using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following standard procedures. RNA purification and DNase digestion were done using an RNA clean and concentrator kit (Zymo Research, Irvine, CA, USA). RNA was quantified using a Qubit RNA broad-range assay kit (Eurogene, OR) on a Qubit fluorometer. The RNA integrity was assessed using the Agilent Bioanalyzer and the Agilent RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). The quality of small RNAs was determined using the Agilent 2100 Bioanalyzer Small RNA kit (Agilent, Santa Clara, CA, USA) The total RNA of each sample was diluted with RNase-free water to obtain a final concentration of 1 mg as a starting material. This diluted total RNA was used to prepare libraries using the protocols by NEXTflex small RNA-Seq kit v3 (Perkin Elmer). Following the protocol from the kit, the 5' and 3' adapters were ligated to the RNA fragments, which were then reverse-transcribed and amplified (18 cycles) to generate cDNA libraries with a unique barcode primer. Libraries were cleaned up using NEXTflex Cleanup beads (gel-free protocol), and the size distribution of the final library was assessed by Agilent Bioanalyzer high-sensitivity DNA assay (Agilent, Santa Clara, CA, USA). The quality-checked libraries were pooled and sequenced in the NextSeq 500 using the single-end 50 bp chemistry at Discovery Life Sciences (Hudson Alpha Institute of Biotechnology, Huntsville, AL, USA).