GSM7102835: Goat number 127 [C_127]; Capra hircus; miRNA-Seq
Milk-derived EVs were obtained by sucrose gradient ultracentrifugations. Then, total RNAs were isolated from EV samples by mirVana Total RNA Isolation Kit (Life Technologies) using Trizol LS Reagent (Ambion) in the firts step. Small RNA-Seq librairies were generated from 100 ng of total RNA using NEBNext library generation kit (New England Biolabs INC.). Sequencing was performed using NewSeq 500 sequencing methodology (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, in the first step, RNA adapters were sequentially ligated to each end of the RNA. Small RNA ligated with 3' and 5' adapters were reverse transcribed and PCR amplified by 15 cycles to create cDNA constructs. Amplified cDNA constructs were size fractionated on a LabChip XT (Caliper Life Sciences) and a band representing adaptors and 15-40 bp insert excised using the manufacturer's instructions. The final cDNA libraries were checked for quality and quantified using capillary electrophoresis. the libraries were pooled in equimolar concentrations. The library pools were finally quantified again with qPCR and optimal concentration of the library pools used to generate the clusters on the surface of a flow cell before sequencing using NexSeq 500 sequencing methodology and reagents, according to the manufacturer instructions (Illumina).