GSM2332214: HF Recovery; Ovis aries; miRNA-Seq
Total RNA were extracted from the frozen homogenized LV tissue using TRIzol® Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. RNA concentration was determined by an ND-1000 Spectrophotometer (Nanodrop™, Wilmington, DE, USA), with OD260/OD280 and OD260/OD230 ratios ³1.90 required. The integrity of RNA was verified using the Bioanalyzer 2100 (Agilent Technology, CA, USA) with RNA integrity numbers (RINs) ≥6.5 required. All RNA samples were stored at −80°C until further processing. Three RNA groups were generated from the pooling of 4 Cont, 6 HF and 4 HF-R samples, and 100 ng of total RNA from each group converted into three small RNA libraries using a NEBNEXT library generation kit (New England Biolabs Inc., Ipswich, MA, USA) according to the manufacturer’s instructions. Each pooled RNA sample was ligated with adaptors to its 3’ and 5’ ends and converted into cDNA before pre-amplification with specific primers containing sample-specific indexes. After 15 cycles, pre-PCR cDNA was purified on QiaQuick columns (Qiagen, Hombrechtikon, Switzerland) and the insert efficiency evaluated by the 2100 Bioanalyzer on high sensitivity DNA chips (Agilent Inc., Waldbronn, Germany). The miRNA cDNA were size fractionated (LabChip XT, Caliper Inc., Hopkintin, MA, USA) and bands representing adaptors and 15-50 base pair (bp) inserts were excised and collected. Based on quality of the inserts and the concentration measurements the 3 libraries were assembled in equal concentrations. The libraries were finally quantified again with qPCR and sequenced on the Illumina NextSeq 500 system (Illumina Inc., San Diago, CA, USA).