RNA-seq of Danio rerio for brain in E3
After extract total RNA and treated with DNase I, Oligo(dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA are fragmented. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for the PCR amplification. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. Then the library is sequenced using Illumina HiSeq 4000 or other sequencer when necessary.