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Old 07-29-2015, 09:43 AM   #1
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Location: Baltimore, MD

Join Date: Jul 2015
Posts: 2
Default Agencourt RNAclean XP bead purification troubleshooting

I recently prepared some low-input rna-seq libraries using NuGEN's Ovation RNA-seq system for model organisms (mouse). After the final step I ran the libraries on a Bioanalyzer and saw that they looked good but had a large population of adaptor dimers present.

Here is a representative image of one of my sample libraries, with a large adaptor dimer peak at ~125bp

I wanted to get rid of the dimers before sequencing, so I did an extra bead purification step using a fresh tube of Ampure RNAclean XP beads that was supplied with the kit (I had previously used the same kind of beads several times during the procedure). I used a bead:sample ratio of 0.8:1.0 in order to remove fragments >200bp to get rid of the adaptor dimers. I followed the protocol exactly including fresh EtOH, optimal drying time, etc. I re-ran the samples on the Bioanalyzer after the purification and I got some very strange results that I'm having trouble interpreting. I have run the post-purification samples twice on the Bioanalyzer with identical results.

Here is a sample of the same library as above, after a bead purification. Note high MW DNA that was previously not present in the sample, as well as a complete lack of most of the DNA that I had seen before. Several of the samples had no detectable DNA other than the upper and lower DNA marker.

Has anyone seen results like these? I'm not sure what could have gone wrong with the bead purification unless I somehow got a bad batch of beads. That still wouldn't explain the new presence of high MW DNA that appeared after purification. I'm hoping it's a problem with the Bioanalyzer but I have no idea what it would be and I haven't found anything during my search of literature and online.

Any help is greatly appreciated, thanks!
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