RNA-seq of human: exosome from differentiated skeletal muscle cells
1) Input RNA is first polyadenylated in order to provide a priming sequence for an oligo(dT) primer. 2) cDNA synthesis is primed by the 3' smRNA dT Primer, which incorporates an adapter sequence at the 5' end of each first-strand cDNA molecule. 3) When the MMLV-derived PrimeScript Reverse Transcriptase (RT) reaches the 5' end of each RNA template, it adds non-templated nucleotides which are bound by the SMART smRNA Oligo-enhanced with locked nucleic acid (LNA) technology for greater sensitivity. 4) PrimeScript RT uses the SMART smRNA Oligo as a template for the addition of a second adapter sequence to the 3' end of each first-strand cDNA molecule. 5) Full-length Illumina adapters are added during PCR amplification. 6) cDNA fragments are sequenced by the read length using sequence by synthesis method on the illumina platform.