We strongly recommend performing a qPCR assay to determine the optimal number of PCR cycles for the library amplification. Information on the qPCR assay can be found in the QuantSeq 3' mRNA-Seq Library Prep Kit User Guide. The PCR Add-on Kit for Illumina (Cat. No. 020.96) includes PCR and Enzyme Mix reagents and P7 primer (7000) that are required for the qPCR assay. In addition, SYBR Green I Nucleic Acid Gel Stain (10,000X in DMSO, Sigma-Aldrich Cat. No. S9430) is also required (user provided).
ATTENTION: The use of SYBR Green qPCR Mastermixes is not recommended and may result in incorrect cycle number calculation.
Typically, libraries prepared from mouse brain total RNA using BC1 Block require one additional PCR cycle compared to libraries made with the standard QuantSeq Kit reagents.