Hi,
I am very new to RNA-seq. I have tried making libraries from 5 samples: 3 from eukaryotic RNA and 2 from bacterial RNA. The starting material was quite similar for the eukaryotic samples in terms of good RIN and good RNA concentration, and for the bacterial samples (low RIN and low concentration) but only one sample of each kind of worked and I don't understand why. I am attaching some BioAnalyzer results for each sample (total RNA, rRNA depleted and purified library). Samples 1-3 are the eukaryotic RNA and samples 4 and 5 are the bacterial RNA. One problem I can see is that I have adapter-dimer contamination in all the samples. I used the AMPure beads, I've read quite a lot about it now here so may try varying the proportion sample:beads although I followed the protocol suggested in the ScripSeq kit.
We thought that the problem could be the fragmentation step, so we fragmented samples 1 and 3 and run them on the BioAnalyzer before and after fragmentation and we can't see anything after fragmentation (see attached file). The big peak at 25 nt is the cDNA primer.
Anyone with experience with this kit have any suggestions? I already tried epicentre tech support.
Many thanks
I am very new to RNA-seq. I have tried making libraries from 5 samples: 3 from eukaryotic RNA and 2 from bacterial RNA. The starting material was quite similar for the eukaryotic samples in terms of good RIN and good RNA concentration, and for the bacterial samples (low RIN and low concentration) but only one sample of each kind of worked and I don't understand why. I am attaching some BioAnalyzer results for each sample (total RNA, rRNA depleted and purified library). Samples 1-3 are the eukaryotic RNA and samples 4 and 5 are the bacterial RNA. One problem I can see is that I have adapter-dimer contamination in all the samples. I used the AMPure beads, I've read quite a lot about it now here so may try varying the proportion sample:beads although I followed the protocol suggested in the ScripSeq kit.
We thought that the problem could be the fragmentation step, so we fragmented samples 1 and 3 and run them on the BioAnalyzer before and after fragmentation and we can't see anything after fragmentation (see attached file). The big peak at 25 nt is the cDNA primer.
Anyone with experience with this kit have any suggestions? I already tried epicentre tech support.
Many thanks
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