normalize Input with Immunoprecipitated raw counts or not

RIP-seq : normalize Input with Immunoprecipitated raw counts or not

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Hi everyone,

I am analyzing a RIP-seq experiment made of 12 RNA libraries as follows :

6 “control” libraries : 3 input (total RNA) and their corresponding immunoprecipitated RNAs (IP) and 6 “affected” libraries : 3 input (total RNA) and their corresponding immunoprecipitated RNAs (IP)

  • Input control 1 – Input control 2 – Input control 3
  • IP control 1 – IP control 2 – IP control 3
  • Input affected 1 – Input affected 2 – Input affected 3
  • IP affected 1 – IP affected 2 – IP affected 3

I would like to analyze on one hand the 3 Input “control” vs the 3 Input “affected” and on the other hand the 3 control IP vs the 3 affected IP.

I am starting with a single raw count table of the 12 libraries. My question is :
Should I split the table in half at the very beginning, an Input count table and IP count table and then perform all the normalization and DE analysis steps in parallel ?
Or should I keep the 12 libraries in the same count table and perform normalization on the whole ?

I tried both and outputs are different unless I’m mistaken. I can not figure out which is the relevant choice. In my opinion, it is not suitable to compare the 6 Input between them from a count table normalized with the whole 12 libraries.

Thanks for your time and your help !


limma


edgeR


analysis


RIP-seq


RNA-seq


DE


table


count


Raw


normalization

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