I extracted total RNA from drug and vehicle treated primary neurons (mouse) and used Kapa Stranded mRNA-Seq kit to generate libraries.
Goal is differential expression analysis - primarily looking at roughly 60 neuronal genes and also a more general effect of our drugs on transcriptional output of neuronal genes.
Input RNA: 1.5ug, PCR cycles - 8x - RNA RIN was always over 8 with good electropherogram trace
Sequencing info: Illumina HiSeq2100 - 5 libraries multiplexed into 1 lane.
So the problem: between 55-60% duplication rate for all libraries - very consistent across the board. The highest number of duplicates are from poly-A and poly-T tracts according to QC data from the sequencing core.
I could really use some advice here. Is this rate of duplication a problem for a DE experiment such as this? What rate of duplication would be more acceptable?
Thanks so much for any input, I'm really worried that my whole PhD project is toast...
Goal is differential expression analysis - primarily looking at roughly 60 neuronal genes and also a more general effect of our drugs on transcriptional output of neuronal genes.
Input RNA: 1.5ug, PCR cycles - 8x - RNA RIN was always over 8 with good electropherogram trace
Sequencing info: Illumina HiSeq2100 - 5 libraries multiplexed into 1 lane.
So the problem: between 55-60% duplication rate for all libraries - very consistent across the board. The highest number of duplicates are from poly-A and poly-T tracts according to QC data from the sequencing core.
I could really use some advice here. Is this rate of duplication a problem for a DE experiment such as this? What rate of duplication would be more acceptable?
Thanks so much for any input, I'm really worried that my whole PhD project is toast...
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