rRNA depletion of RIP-seq samples

Question

[CrisRisu]

Hi, I am new to RIP-seq experiments, and I have a question about the type of library to use. I have seen that it is possible to make libraries with or without rRNA depletion and I don't know what would be optimal for my experiment. Looking for information I have read that if the RBP is not expected to bind rRNA, it is advisable to deplete ribosomal RNA (rRNA) from all RNA samples using Ribo-Zero (epicentre) prior to generating libraries. In principle, my protein does not bind rRNA although I cannot rule it out. Taking into account that the isolated RNA is immunoprecipitated, is it very critical to use or not the rRNA depletion?

I would appreciate any advise or information. Thank you in advance

[dsull]

This is not really a bioinformatics question. But can't you check if you have an unsubstantial amount of rRNA (or not) post-immunoprecipitation on an agilent bioanalyzer?

[GenoMax]

Please post at seqanswers.com or biology stackexchange.

[noodle]

The concern with rRNA is that it will be ~90% of reads unless you can separate what you're after from 'the ribosome'. Separation comes in 2 varieties, enrichment or depletion, but seldom both. If you can enrich the RNA you're after without also targeting rRNA with your binding protein/antibody, depletion should not be needed. Any commercially sold product should have accompanying literature with raw data provided and you can computationally investigate how much rRNA comes through to the end library (hint: it will never be zero).