GSM9109244: Nor_GFP_1; Homo sapiens; miRNA-Seq
Trizol The RNA isolated from each sample was used to construct sequencing libraries with the SMARTer smRNA-Seq Kit for Illumina, following the manufacturer's protocol. Briefly, Input RNA is first polyadenylated in order to provide a priming sequence for an oligo-(dT) primer. 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. When the MMLV-derived PrimeScript™ Reverse Transcriptase (RT) reaches the 5' end of each RNA template, it adds nontemplated nucleotides which are bound by the SMART smRNA Oligo-enhanced with locked nucleic acid (LNA) technology for greater sensitivity. In the template-switching step, 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. In the next step, full-length Illumina adapters (including index sequences for sample multiplexing) are added during PCR amplification. The Forward PCR Primer binds to the sequence added by the SMART smRNA Oligo, while the Reverse PCR Primer binds to the sequence added by the 3' smRNA dT Primer. Resulting library cDNA molecules include sequences required for clustering on an Illumina flow cell.