mRNA library preparation Adapter ligation

Question

[imran]

We are starting making our own library for mRNA sequencing, and we are totally clueless about what adapter sequence to use! I have gone through the Illumina adapter sequences on their websites, but there are larger sequences then what my kit provider (Vazyme) is offering. Any one can tell me about how to start with adapter sequences and what adapter sequence to add, we will be using Novaseq 6000 machine and PE150 mode Thanks

[jkim]

Before preparing your libraries, there are some QC steps for sequencing cores to ensure that your samples are suitable for sequencing. For example, one of the criteria is the RIN score. I recommend reaching out to a sequencing core, vendors, or Illumina to confirm that the quality of your samples meets their QC criteria.

Answer 1

[GenoMax]

You will need to check which exact kit you are using but this is one example of adapter sequences available from Vazyme. This is what you will need to use when trimming data for this kit.

Answer 2

[ATpoint]

This is wetlab and by this off-topic. Please contact the manufacturer for advise. It depends on how exactly fragments were made (e.g. enzymatic/tagmentation vs fragmentation), so there is no easy answer to this. Ask them, they must know.

Actually, it gets increasingly uncommon to make your own libraries these days. If you haven't started yet consider to send RNA directly to a sequencing center and let them do the full library preparation, QC and sequencing. That is often cheaper than buying everything you need yourself.