What is the expected size distribution of double-stranded cDNA generated by SMARTer cDNA synthesis kits?

SMARTer Ultra low kits: Successful cDNA synthesis and amplification should produce a cDNA library spanning 400–9,000 bp. The main peak should occur at approximately 2,000 bp.SMARTer stranded kits: Successful cDNA synthesis and amplification with the SMARTer Stranded RNA-Seq Kit should yield a distinct Bioanalyzer electropherogram peak spanning 150–1,000 bp, centered on approximately 300 bp. When [...]

How do I analyze double-stranded (ds) cDNA quality?

We recommend analyzing double-stranded (ds) cDNA generated with SMARTer kits using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Chip (Agilent, Cat. # 5067-4626) before sequencing.Prior to cDNA library analysis, ensure that the electropherogram of the High Sensitivity DNA Ladder is properly displayed: showing a flat baseline, well-resolved ladder peaks, and properly identified Lower [...]

Can I collect cells directly in Reaction Buffer?

If necessary, cells may be collected directly in Reaction Buffer containing RNase Inhibitor, followed immediately by cDNA synthesis or freezing.Note: If cells are collected and frozen in Reaction Buffer, add fresh RNase Inhibitor after thawing cells and prior to cDNA synthesis. General [...]

Can I freeze collected cells prior to cDNA synthesis?

If you cannot immediately proceed with cDNA synthesis, you may freeze cells on dry ice and store at –80°C. Gently centrifuge cells, remove the collection medium and freeze the cell pellets. Collected cells may also be frozen in media compatible with the SMARTer Ultra low protocol. (See ""What media have been tested for compatibility with direct cDNA synthesis from intact mammalian cells?"") [...]

What is the recommended volume of Reaction Buffer for various amounts of cells when using SMARTer Ultra Low kits?

For cell lysis using the SMART-Seq mRNA Kit for Sequencing, we recommend the addition of 1 µl of 10X Reaction Buffer followed by a 5-minute incubation at room temperature. For the legacy kits, the amount of Reaction Buffer will vary depending on the input volume of your RNA or cell sample. Maintaining the same volume of Reaction Buffer for all cell samples is not necessary. Do not add more than [...]