Dear community,
I need some help, as RNA-sequencing is quite new for me...
I prepared DNA libraries from RNA for sequencing with illumina. I analyzed the quality of the libraries on the bioanalyzer afterwards revealing that I had a remarkable amount of too big fragments in them (500 bp and higher holding 25 % of the whole library), also the peak around 250 bp decreased quite slowly... I read it might have been a problem of bubble product resulting from the final PCR step of the library preparation (low primer efficiency, too many cycles...).
Fair enough, as I don't have too much of my samples, I decided to run a gel with my libraries and to cut out just all the big fragments. That turned out fine exept that - after clean up with Qiagen gel extraction kit - I have super low ratios of 260/230 (around 0,1) and super high ratios of 260/280 (around 6). There is a high peak at about 240 nm. I read it might be some salt from the buffers or acetate. In the meantime I found some good advice here to avoid it.
My question is, does the salt/acetate matter for sequencing? In another thread here Qiagen says it doesn't at least for qPCR. The yield after the gelextraction is absolutely fine... So has anybody got any experience concerning that?
I will be most thankful if you'd shared your thoughts with me!
I need some help, as RNA-sequencing is quite new for me...
I prepared DNA libraries from RNA for sequencing with illumina. I analyzed the quality of the libraries on the bioanalyzer afterwards revealing that I had a remarkable amount of too big fragments in them (500 bp and higher holding 25 % of the whole library), also the peak around 250 bp decreased quite slowly... I read it might have been a problem of bubble product resulting from the final PCR step of the library preparation (low primer efficiency, too many cycles...).
Fair enough, as I don't have too much of my samples, I decided to run a gel with my libraries and to cut out just all the big fragments. That turned out fine exept that - after clean up with Qiagen gel extraction kit - I have super low ratios of 260/230 (around 0,1) and super high ratios of 260/280 (around 6). There is a high peak at about 240 nm. I read it might be some salt from the buffers or acetate. In the meantime I found some good advice here to avoid it.
My question is, does the salt/acetate matter for sequencing? In another thread here Qiagen says it doesn't at least for qPCR. The yield after the gelextraction is absolutely fine... So has anybody got any experience concerning that?
I will be most thankful if you'd shared your thoughts with me!