Can I use a pooling option for the SMART-Seq Stranded cDNAs, generated from bulk RNA inputs?"

No, one can pool only SMART-Seq Stranded cDNAs generated from single cells. SMART-Seq Stranded pooling is beneficial for single cells, since cell to cell variation is lower than that between bulk RNAs of variable quality. cDNAs generated from bulk RNAs of variable quality will have variable cDNA yield and size distribution, that is not feasible to predict or control.SMART-Seq Stranded [...]

What RNA fragmentation method is used in the SMART-Seq Stranded workflow?

SMART-Seq Stranded protocol is using RNA heat fragmentation in the presence of Mg++ included in the scRT Buffer: fragmentation is stopped by placing the samples on ice for 2 min.SMART-Seq Stranded [...]

Will there be genomic DNA contamination in the SMART-Seq Stranded libraries, generated from whole cells?

Current SMART-Seq Stranded cell lysis and fragmentation conditions, using as input whole intact cells, minimize potential genomic DNA contamination.SMART-Seq Stranded [...]

What RNA purification kits are recommended for compatibility with the SMART-Seq Stranded cDNA synthesis?

We recommend the NucleoSpin RNA XS kit offering on-column DNAse I treatment as well as an optional DNAse I treatment of RNA eluate, followed by column purification. To ensure a complete removal of DNA, essential for the highly sensitive SMART-Seq Stranded cDNA synthesis, it is advisable to perform an additional DNAse I treatment of purified RNA eluate, since single-stranded DNA (present in [...]

Is the SMART-Seq Stranded Kit compatible with LCM and FFPE/ PFA cells?

SMART-Seq Stranded workflow is not compatible with cDNA synthesis directly from FFPE/ PFA or LCM cells. Fixed cells may contain high level of single-stranded DNA which can be randomly primed and used as a template by the reverse transcriptase, thus incorporating genomic DNA sequences into the final SMART-Seq Stranded [...]