How to normalise Nanopore mRNA sequencing data between two cell lines

Question

[Mo]

Hi,

I have a GFP library (plasmid DNA) that I have transfected in two different cell lines and sequenced mRNA on Nanopore. After sequencing, I align data with GFP sequences and count the reads for each GFP variant for each cell line. This is my raw GFP mRNA data.

To normalise the raw mRNA seq data, I have also sequenced the GFP library (plasmid DNA) without any PCR on Nanopore and quantified the abundance of each GFP variant as DNA in the library. Here, after sequencing I have aligned the data with the GFP reference file and counted reads for each GFP. This is my GFP DNA data.

To normalise raw mRNA, i divide mRNA data by DNA data for each GFP variant. This is my normalised mRNA.

I have this data for two cell lines. Now I want compare normalised mRNA seq data between these two cell lines.

How can I do that?

Thanks.

[lagartija]

It's not very clear to me. Why would you normalize on the GFP DNA ? It is a way to normalize on cell number ? Actually why not

But what is your question ? How to find differentially expressed genes ?