title: GSM4043017: Dermal papilla-derived exosomes, P8-Exos1; Homo sapiens; miRNA-Seq
library_construction_protocol: Media were collected from human dermal papilla cultures (P3 or P8) and stored at -20 °C. Exosomes were isolated using ultracentrifugation (SW 32 Ti swinging-bucket rotor in a Beckman Coulter L8-80M Ultracentrifuge), as described previously.[26] Briefly, 500 ml hDPC culture medium was centrifuged at 2000 × g for 20 minutes, then at 10,000 × g for 20 minutes, to remove cells and debris. The supernatant was filtered through a 0.2 µm syringe filter (Corning, Germany), and ultracentrifuged at 100,000 × g for 60 minutes at 4 °C twice. The final pellet was suspended in 100–200 µl PBS and stored at -80 °C. Libraries for miRNA sequencing were generated using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (NEB,USA) according to the manufacturer's instructions. 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.
attributes.tag: GEO Accession
attributes.value: GSM4043017