Is SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian compatible with cDNA synthesis directly from cells?

SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian has been validated with 50-1,000 whole intact cells. Damaged cells usually contain high quantities of single-stranded DNA which can be randomly primed and used as a template by the SMARTScribe™ Reverse Transcriptase, thus incorporating genomic DNA sequences into the final SMARTer Stranded v2 Pico or cDNA library. Damaged cells are [...]

What RNA purification kits are recommended for compatibility with the SMARTer Stranded v2 - Pico cDNA synthesis?

We recommend the NucleoSpin RNA XS and NucleoSpin totalRNA FFPE XS kits offering on-column DNAse I treatment as well as optional DNAse I treatment of RNA eluate, followed by column purification . To ensure a complete removal of DNA, essential for the highly sensitive SMARTer Stranded Total RNA-Seq Kit v2 - Pico cDNA synthesis, it is advisable (if feasible) to perform an additional DNAse I [...]

Is SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian compatible with RNA purified from FFPE/PFA or LCM cells?

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is compatible with purified RNA obtained from FFPE, PFA-fixed, and LCM cells, that does not contain any remnants of DNA, carrier or co-precipitant. RNA from FFPE or PFA-fixed cells must be decrosslinked and thoroughly treated with DNAse in order to remove all traces of DNA, since single-stranded DNA (often present in FFPE, [...]

If RNA fragmentation is not required, what is the purpose of heating RNA at 72oC?

Incubation at 72oC unwinds RNA secondary structure, allowing random primer annealing, performed in the absence of the 5X First -Strand Buffer (containing Mg++) to avoid RNA fragmentation.Stranded RNA-seq for mammalian [...]

What is the mechanism of the RNA fragmentation method used in the workflow of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian?

SMARTer Stranded v2 Pico protocol is using RNA heat fragmentation in the presence of Mg++ included in the 5X First -Strand Buffer: fragmentation is stopped by placing the reaction mix on ice for 2 minutes.Stranded RNA-seq for mammalian [...]