I am optimizing conditions for an RNA-IP Sequencing (RIP-seq) experiment. The goal is to identify associated RNAs that co-IP with a particular transcript.
For my experimental samples and positive control, I am getting about 10-fold more RNA pulled down than for the negative control (as measured by Qubit). An initial screen with RT-qPCR shows that my transcript of interest is much more enriched in the samples than the negative control (great news).
My concern is the difference in total RNA pulled down in the IP. Will the discrepancy between RNA concentrations affect downstream steps in library preparation for RNA-seq? Is it ok to use a negative control for RIP-seq that pulls down significantly less RNA than the experimental samples? Do I need to find a negative that pulls down the same total concentration of RNA but only fails to pull down my transcript of interest?
Any advice would be appreciated. Thanks!