Library eluted with TE buffer (low EDTA)?

[Iva]

Hi all,

I'm doing my first RNAseq experiment. I followed the NuGen Ovation Ultralow Library V2 protocol which suggests throughout to elute after every bead purification step in TE (low EDTA). This is also what they suggest for the final elution of amplified library. Obviously, they don't see a problem with 0.1mM EDTA in the library (my reasoning is that it will inevitably be further diluted when pooling the libraries and won't pose such an issue?). However, I only read our genomics facility requirements now and I see that they do not want any EDTA in the libraries, to prevent the sequencing reaction failure. Do you think I should re-purify my libraries with beads (if so, in what ratio) and elute in the non-EDTA containing buffer? Molarities of my libraries are quite variable - from 6 nM up to 30 nM, so I am afraid of losing a lot if I re-purify.

Thank you!

[jdk787]

I don't think the EDTA should be a problem, but if your facility insists you can use a 1.5X Ampure bead cleanup to swap out the buffer. If you are pooling your libraries you can just cleanup the pool.

[nucacidhunter]

Low TE buffer is fine and I have not seen any adverse effect on sequencing in comparison with EB or Illumina resuspension buffer.

Your genomic facility will not know your buffer composition.

[Iva]

Thank you for your answers and help! I ended up pooling everything and cleaning up the pool with MinElute reaction cleanup. Worked like a charm.