How should sequencing libraries be prepared from cDNA generated with SMARTer Ultra low kits?

Illumina indexes and adapters are integrated during cDNA amplification with the SMARTer Stranded RNA-Seq Kit (including the HT version) and the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian. No additional library preparation is needed. (see ""Which indexes are included in SMARTer stranded kits?""). Not all indexes can be pooled together, consult the Illumina literature (such as the [...]

Can I analyze unpurified double-stranded (ds) cDNA for PCR cycle optimization?

PCR-amplified ds cDNA can be analyzed directly from the PCR reaction mix, prior to SPRI bead purification, using an Agilent 2100 Bioanalyzer. The ds cDNA profile will contain a large peak immediately following the Lower Marker; this represents the primer or primer dimers. The Bioanalyzer software may assign the primer/primer-dimer peak as the Lower Marker. If this occurs, manually reassign the [...]

What can I do if I have low cDNA yield?

If the ds cDNA yield is less than 2 ng, you may further amplify the ds cDNA using several additional PCR cycles, based on the ds cDNA concentration (determined by Agilent 2100 Expert Software). Continue to avoid overcycling. It is preferable to use too few cycles rather than too many.An electropherogram trace of low concentration ds cDNA generated with a SMARTer Ultra low kit. The red arrow [...]

How do I determine the double-stranded (ds) cDNA yield

Estimate the yield of ds cDNA using an Agilent 2100 Bioanalyzer: Open the ds cDNA electropherogram using Agilent 2100 Expert Software. Choose the ""Region Table"" tab. Select the expected size range of ds cDNA appropriate for your SMARTer kit. The ds cDNA concentration will be displayed below the graph. For SMARTer Ultra low kits: select the region encompassing 400–9,000 bp. For the SMARTer [...]

What is the expected double-stranded (ds) cDNA yield?

SMARTer Ultra low kits The cDNA yield in newer generations of SMARTer Ultra low kits is higher compared to that of the legacy kits. Please refer to the user manual of your specific kit for more details. In general, depending on the RNA source, integrity, input amount, and the final volume of the library, the expected yield of ds cDNA generated using SMARTer Ultra low kits is 2–17 ng. This is [...]