I am preparing RNA-seq libraries from bacterial samples unsing NEBNext Ultra II Directional Library Kit. I prepared the samples and I did Ampure Bead purification second time with the samples I see primer and adaptor dimers. Some of my samples did not get purified and remove adaptor dimers, infact I observed this shoulder peak or double peak in my target library. How I can remove that? Would it cause a problem in the library? I have included the images from tapestation.
I am currently having the very same issue. It happens when there is low input RNA, or low quality of the input RNA.
You can perform an additional Ampure cleanup. I have just done this, reducing the ratio to 0.9X, and have successfully saved my libraries. The next step in my protocol is a capture step, and I am told that the dimer would not have persisted through the capture, but I'd rather get rid of it beforehand.
0.9X volume of Ampure beads will remove anything that is less than 175bp from your library.
I repeated Ampure Beads cleanup with 1X and diluted samples before loading tapestation bc previous ones were not diluted and I read somewhere not diluted samples might create that shoulder peak. Now the libraries seem ok but the problem is having very low intensity.
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