Diluting Nextera XT Indexes?

[Amygdala]

Hey all,

My samples originate from LCM, and I am currently following the SMART-seq2 workflow. Recently one group has combined both these approaches and written a nice protocol for it: https://doi.org/10.1007/978-1-4939-7213-5_6

Both Picelli's SMART-seq2 protocol (for single cells) and the above LCM-seq protocols mentions diluting the index adapters from the Nextera XT Index Kit, which appears to go against Illumina's protocol for adding 5ul of each adapter per sample without diluting. In the Picelli's original paper the authors do not dilute the index adapters, so I am a bit confused.

Can anyone clear this up for me? Or explain the benefit of diluting the adapters? I would have thought that since I am adding the recommended 1ng of input material for the Nextera XT library prep kit that I would follow Illumina's protocol of not diluting the adapters.

Many thanks!

[kmkocot]

I've also been curious about this. We often have a leftover index/adapter peak in our fragment analyzer traces of Nextera XT libraries made from SMART-Seq HT cDNA. I don't have a helpful answer but it seems like a reasonable thing to do. We struggle to get ideal Nextera XT library peaks following the Illumina protocol closely so we have been scared to change the amount of adapter used, though.

[aurman]

While I do not have any advice for your question, I was wondering if you knew the initial concentration of the Nextera XT indices. I ordered custom adaptors, but am not sure what concentration to add.