Hi there,
I would like to ask whether someone has ever experienced a similar problem with hexamer primed cDNA libraries?
We have prepared a cDNA library starting with high quality RNA from mice (RIN = 9). In the final QC, we loaded a fraction of the lib onto a fragment analyzer. As you can see in the attached pic, the lib resembles a hedgehog with peaks every 10bp. When we sequenced the lib, we found that the peaks consisted of a decamer (GTATATACTA). Sometime up to 40 of these decamers are present. Thus, it´s not an issue of the fragment analyzer.
We have really no idea what this could be. We see that for different samples, but it´s not consistent. Some days the lib looks perfect, the other day we get this shape.
I´m really looking forward to your opinions!
I would like to ask whether someone has ever experienced a similar problem with hexamer primed cDNA libraries?
We have prepared a cDNA library starting with high quality RNA from mice (RIN = 9). In the final QC, we loaded a fraction of the lib onto a fragment analyzer. As you can see in the attached pic, the lib resembles a hedgehog with peaks every 10bp. When we sequenced the lib, we found that the peaks consisted of a decamer (GTATATACTA). Sometime up to 40 of these decamers are present. Thus, it´s not an issue of the fragment analyzer.
We have really no idea what this could be. We see that for different samples, but it´s not consistent. Some days the lib looks perfect, the other day we get this shape.
I´m really looking forward to your opinions!
Comment