[ScienceGrrl]

Hello Arthur45,

Make sure you have a very accurate quant of your input samples. Nextera is a mass driven setup so if you are off on your initial quant at all, can get results of too large/too small fragments very easily.

[Simone78]

hi Arthur 45,
sorry for the misunderstanding. You do need to clean up the cDNA after the first PCR (pre-amplification for the Smart-seq2 protocol). I am not really familiar with the latest version of the Nextera protocol but the 30 + 30 ul beads sounds a bit strange to me.
I know that Nextera is using the Zymo and Nextera XT is not. We decided to not use a column purifcation because it´s cheap and equally effective.
That said, I am not sure what you have there. A single fragment at 1 kb doesn´t look like a standard library (is it a ladder or similar?). Why don´t you just take some tot RNA , let´s say 1 ng, do some pre-ampl and try to tagment that? I agree that the Tape after tagmentation has no library. Don´t really know why, though

[Arthur45]

Hi both,

thanks for your answers

About the 30+30 ul steps: I called Illumina since and the representative agreed that this *might* be a typo. He raised an issue for their protocol team. This team will change the protocol when the next version of the protocol will be out.

About my graphs: the graph with the 1kb peaks is my DNA before starting the library. The other graph is right after the tagmentation. It looks like there is no more DNA after tagmentation. I am starting to think that my input is not of the right concentration like ScienceGrrl said. And also, I read somewhere that DNA should be clean before starting a library, so I will try to clean my phenol-chloroform extractions with a spin column, just in case.

Cheers,
arthur