Has anyone been using the TruSeq RNA Access kit? We have been getting a snoRNA (SNORA63) soak up almost 10% of our reads from libraries we've generated with this kit. Seems to be present if we use high or low quality starting material. I'm attaching our Bioanalyzer traces, and you can clearly see there is an over-represented transcript present. I'm wondering if anyone else is having a similar issue? Any thoughts on whether this is due to insufficient post-capture washing? Thanks!
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We have seen several different snoRNA's soak up reads in our analysis. We have been seeing the data improve recently by pre-heating the wash buffer during the second wash following captures. I can't say with absolute certainty that this will fix your problems. I think part of this problem begins with sample quality/complexity. Are you using FFPE material?
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Unfortunately, I don't have any head to head comparison data to share. The snoRNAs are actually targeted by probes within the RNA Access oligo pool, so in many cases there is nothing you can do do get rid of them. We implemented the heated wash step because we figured it couldn't hurt. The workflow generates such high exonic mapping rates that we simply stopped worrying about the contaminating snoRNAs. At the end of the day, their presence wasn't dramatically affecting our work.
What alignment rates are you seeing for snoRNA's in your experiment?
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