Hi there Can anyone explain to me how to use the ESTIMATE package in RNA-seq analysis? I want to calculate immune scores and stromal scores by employing the ESTIMATE algorithm, then analyze the relationship of immune/stromal scores with subtype classification and cytogenetic risk by one-way analysis of variance, but I don't know how to do this!

I will be grateful for any help you can provide.

Can you please elaborate on what you have already tried and on which part (or parts) you are having trouble? Thank you.

How can I use this package? I want to know whether I should normalize data before use or not? And at last, how can I read the .gct output file?

How to prepare the .gct file to use in DESeq2