SriptSeq library preparation

[airun78]

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

[vadoue]

Hi,

I am happy to read you since I have exactly the same problem... I used others library kits before and I never got any primer-dimer contamination. This time I used ScriptSeq and I have an extra peak at exactly 136 bp in all my samples (and maybe another at 172 bp but I can't really see it due to the overlap with my library fragments). I am surprized since usually people say that primer dimer contamination is 120 bp. Is you extra peak also at 136 bp? I will interested to see any reply here... Good luck!

[vadoue]

It seems that a second Ampure purification step (1:1) worked for me to removed most primer-dimers. You should use less adaptors in your protocol. In your bioanalyser profile, the contamination is so huge that you can't even see your library profile (which could be good). If you use a new Ampure purification step, be carefull to quantify the exact volume in each tube to have the exact 1:1 ratio with beads to remove specifically everything below 200bp. Good luck!

[airun78]

Thanks vadoue, I'll try a second Ampure purification step.