Workflow overview for the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian.
Schematic of technology in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. SMART technology is used in this ligation-free protocol to preserve strand-of-origin information. Random priming (represented as the green N6 Primer) allows the generation of cDNA from all RNA fragments in the sample, including rRNA. When the SMARTScribe Reverse Transcriptase (RT) reaches the 5' end of the RNA fragment, the enzyme’s terminal transferase activity adds a few non-templated nucleotides to the 3' end of the cDNA (shown as Xs). The carefully designed Pico v2 SMART Adapter (included in the SMART TSO Mix v2) base-pairs with the non-templated nucleotide stretch, creating an extended template to enable the RT to continue replicating to the end of the oligonucleotide. The resulting cDNA contains sequences derived from the random primer and the Pico v2 SMART Adapter used in the reverse transcription reaction. In the next step, a first round of PCR amplification (PCR1) adds full-length Illumina adapters, including barcodes. The 5' PCR Primer binds to the Pico v2 SMART Adapter sequence (light purple), while the 3' PCR Primer binds to sequence associated with the random primer (green). The ribosomal cDNA (originating from rRNA) is then cleaved by ZapR v2 in the presence of the mammalian-specific R-Probes v2. This process leaves the library fragments originating from non-rRNA molecules untouched, with priming sites available on both 5' and 3' ends for further PCR amplification. These fragments are enriched via a second round of PCR amplification (PCR2) using primers universal to all libraries. The final library contains sequences allowing clustering on any Illumina flow cell.