GSM5388478: empty3D2; Homo sapiens; RNA-Seq
Total mRNAs from DHT2D, DHT3D, empty2D, and empty3D were extracted and subjected to miRNA sequencing by TRIzol reagent (Invitrogen, Gaithersburg, USA) per manufacturer instructions. Libraries for mRNA sequencing were generated using the NEBNext Multiplex 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.