GSM4905048: pa4; Homo sapiens; miRNA-Seq
Exosomes were separated via ultracentrifugation RNA degradation and contamination, especially DNA contamination, was monitored on 1.5% agarose gels. RNA concentration and purity were measured using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). A total amount of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextR UltraTM small RNA Sample Library Prep Kit for IlluminaR(NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, first of all, ligated the 3′ SR Adaptor. Mixed 3′ SR Adaptor for Illumina, RNA and Nuclease-Free Water, mixture system after incubation for 2 minutes at 70 degrees in a preheated thermal cycler. Tube was transfer to ice. Then, add 3′ Ligation Reaction Buffer (2X) and 3′ Ligation Enzyme Mix ligate the 3′ SR Adaptor. Incubated for 1 hour at 25°C in a thermal cycler. To prevent adaptor-dimer formation, the SR RT Primer hybridizes to the excess of 3′ SR Adaptor (that remains free after the 3′ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated. The second, ligated the 5′ SR Adaptor. Then, reverse transcription synthetic first chain. Last, PCR amplification and Size Selection. PAGE gel was used to electrophoresis fragment screening purposes, rubber cutting recycling as the pieces get small RNA libraries. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.