Workflow was as follows:
1) Extracted mRNA from Ribotag animals, converted into libraries using Nugen kits that are optimized for Illumina sequencers.
2) Quality control checks (bioanalyzer, qPCR) show that library size distribution, quality and concentration are are correct (i.e. avg 350 bp, diluted to 10 nM for sequencing at 10 pM).
However, for some reason, my libraries won't cluster properly. We send our samples to an outside company, but we've used them before and never had any problems. Anyone have any experience with inexplicable low cluster densities with RNA-seq?
1) Extracted mRNA from Ribotag animals, converted into libraries using Nugen kits that are optimized for Illumina sequencers.
2) Quality control checks (bioanalyzer, qPCR) show that library size distribution, quality and concentration are are correct (i.e. avg 350 bp, diluted to 10 nM for sequencing at 10 pM).
However, for some reason, my libraries won't cluster properly. We send our samples to an outside company, but we've used them before and never had any problems. Anyone have any experience with inexplicable low cluster densities with RNA-seq?