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 [...]

What is the expected size range of fragmented, ds cDNA after library preparation with Nextera kits?

The Nextera kits from Illumina produce libraries with a size range of 300–1,000 bp. Please refer to the Nextera DNA Sample Preparation Guide or Nextera XT DNA Sample Preparation Guide for more specific details.General [...]

Do I have to scale down the Nextera XT DNA Sample Preparation Kit protocol when using 100–150 pg of ds cDNA?

No. Use 100–150 pg of ds cDNA generated with the SMARTer Ultra low kit in the input volume recommended in the Nextera XT DNA Sample Preparation Guide. Follow the rest of the protocol as written.General [...]

Can I use more than 150 pg of ds cDNA for the Nextera XT DNA Sample Preparation Kit?

In our hands, using 100–150 pg of input cDNA with the Nextera XT DNA Sample Preparation Kit generates DNA fragments with an optimal average size for Illumina cluster generation and sequencing. Using more than 150 pg of ds cDNA is not recommended since it generates significantly larger DNA fragments, which are suboptimal for Illumina cluster generation and sequencing. For a greater cDNA input [...]

How do you explain the presence of introns in the final SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian cDNA library?

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian uses a random priming strategy that generates cDNA from coding, noncoding, and alternatively spliced RNAs that may carry introns. Additionally, previously unannotated transcripts may be assigned as introns by your software's default.General [...]