Why should I perform a positive-control cDNA synthesis reaction?

A positive-control cDNA synthesis reaction, using control RNA included in each SMARTer kit, enables verification of kit performance and components and helps in evaluation of your sample cDNA library.Tips for preparing the control reactions: Prepare fresh dilutions of the Control RNA. Do not use previously diluted low-concentration RNA samples, since RNA is less stable at low concentrations. If [...]

Why do library concentrations obtained with a qPCR-based method differ from those obtained by other methods?

qPCR only measures the library molecules that can be used for cluster generation. Other methods cannot differentiate between DNA molecules with or without adaptors, resulting in inaccurate quantification of the functional fraction of the library.General [...]

Why is quantification of NGS libraries by qPCR better than using other methods?

By using qPCR primers that anneal to the sequencing adaptors, you can quantify just the fraction of the library capable of cluster generation. qPCR is also extremely sensitive, consuming only a small amount of your sample and making it ideal for accurate quantification of very dilute libraries.General [...]

Why should I quantify my libraries prior to sequencing?

To obtain the highest quality NGS data, loading the flow cell with an appropriate amount of library DNA is essential. An insufficient amount of library DNA will generate low-density clusters and reduced sequencing yield. Excessive amounts of library DNA may increase cluster density, resulting in poor data quality. In addition, for multiplexed sequencing, there must be an equal amount of each [...]

How should sequencing libraries be prepared using SMARTer stranded 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 [...]