Question: RIP-Seq data analysis
1
gravatar for flyguymail22
11 months ago by
flyguymail2210
flyguymail2210 wrote:

Hi

I have performed a RIP-Seq experiment, that is RNA Immunoprecipitation followed by RNA sequencing. Does anyone know of an established pipeline or tool as to how to analyse this data?

Thank You. FlyGuy

sequencing rna-seq ip peak • 847 views
ADD COMMENTlink modified 27 days ago by Raheleh130 • written 11 months ago by flyguymail2210
1

enter image description here

Tried or searched for anything yet, e.g. via the search function?

ADD REPLYlink modified 11 months ago • written 11 months ago by ATpoint29k

I've got the RIP-seq analysis outputs. We used an isotype control antibody VS antibody against an RBP to pull down corresponding RNAs!

I did the following pipeline: Hisat2 (mapping the reads after QC) -> htseq-count (getting read counts) -> DESeq2 (getting enriched RNAs in test VS isotype control) -> volcano plot shows up-regulated and down-regulated genes in the test sample.

Logically, I should have seen just enriched RNAs (up-regulated genes), but surprisingly there are some RNAs down-regulated in the test sample (or up-regulated in the isotype control sample)!!!!! How can we explain and interpret this???? it seems some genes are pull down in isotype control sample as well. Are these background? Is my computational pipeline right?

BTW, it's worth to mention that all enriched RNA (called up-regulated genes in DESeq2 pipeline) are significantly relevant and meaningful to my experiment. However, I am not sure how much confident I can be on this output? And how should I explain down-regulated RNAs? because normally we just expect enriched RNAs in RIP-seq experiment.

ADD REPLYlink written 27 days ago by Raheleh130

Is there any public data can be used as example dataset to develop RIP-seq analysis pipeline?

ADD REPLYlink written 27 days ago by Shicheng Guo8.0k
1

RIPseq data available if you search via SRA-explorer.

ADD REPLYlink modified 27 days ago • written 27 days ago by genomax78k
2
gravatar for Konstantinos Yeles
6 months ago by
Italy
Konstantinos Yeles100 wrote:

You can use RIPSeeker package from Bioconductor, or you could perform a "differential enrichment" analysis with edgeR / DESeq2 packages.

ADD COMMENTlink written 6 months ago by Konstantinos Yeles100
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1734 users visited in the last hour