GSM2575753: VIR rep2; Bos taurus; miRNA-Seq
Total RNA of Theileria-infected leukocytes was isolated using the miRNA isolation kit (Thermo fisher scientific #AM1560) according to the manufacturer’s instructions. The quality and quantity of RNA was measured by Nanodrop spectrophotometer. Total RNA designated for miRNA experiments was extracted using the mirVana miRNA isolation kit (Thermo Fisher) using the manufacturer’s instructions.  The quantity and quality checked by Qubit and Bioanalyzer 2100, respectively. The miRNA libraries were prepared using the Truseq Small RNA Sample Preparation kit following the manufacturer’s instructions. Briefly, 1 μg of total RNA was adapter ligated at the 3’ and 5’ ends, reverse transcribed, barcoded then amplified with 11 PCR reactions. Then the cDNA was run on a 6% TBE PAGE gel (Novex, Life Technologies). After staining with SYBR Green the gel is visualized on a UV transluminator (Doc-It imaging system, UVP) and the cDNA constructs of a size between 145-160 bp were cut out and eluted from the gel, concentrated and the libraries validated, quantified and finally pooled.