RNAseq data analysis of pooled data

Question

[luffy]

Hello experts,

Kindly request you share your thoughts and tutorials for RNAseq data analysis from Raw data (FASTQ files) to arrive at differentially expressed genes.

I have brief understanding of theory based on this and this it would really helpfully if anyone could share tutorials/code for the same.

What is the best approach for pooled data? some of the posts here in the forum recommends not using pooled data but in this case i have to. so kindly share your thoughts on how to modify or alter the workflow/code/approach since i am using pooled data

Any help would be much appreciate it

Thank you for your time

[Dunois]

I'd look at the DESeq2 documentation and maybe this tutorial as a start. There are couple of nice tutorials on YouTube also.

[luffy]

Dunois Thank you for the tutorial, what are your thoughts for approach for pooled data? should i follow the documentation/the tutorial or any suggestions for any modification in any step?

[Dunois]

What exactly do you mean by "pooled" data?

[luffy]

Dunois the person who did this experiment said they used qiagen kit to extract RNA (from clones of podocyte cells) and then they pooled patients (2-3 patients) who are from same category (In total we have 5 categories) into one and ribo depletion was carried out, before library preparation step. This is the information given to me, let me know if you need more information regarding this.

Thank you

[Dunois]

So I guess you have 3+ replicates per category and 5 categories, where each replicate represents the material from 2-3 such patients as you mentioned?

[luffy]

Yes correct. but 2 replicates per category and 2 replicates of healthy control

[Dunois]

This is just "regular" RNAseq data then, as far as I can tell. I think you should be able to take pretty much any standard tutorial and adapt the workflow to your data. (People who are much more knowledgeable than me, please do chime in if I'm incorrect.)

[ponganta]

By "pooled" data, do you mean data from tissue-specific bulk-RNAseq? Or did you pool single cell (SC) RNAseq-libraries?

[luffy]

@ponganta, I spoke to the person who had done this experiment, they used qiagen kit to extract RNA (from clones of podocyte cells) and then they pooled patients (2-3 patients) who are from same category (In total we have 5 categories) into one and ribo depletion was carried out, before library preparation step. This is the information given to me, let me know if you need more information regarding this.

Thank you

Answer 1

[grant.hovhannisyan]

I made a tutorial last year for University students on this topic https://www.loom.com/share/a55c1dedb8e64e7cac47f6595b751de7. Let me know if you also want to get the data and codes of this course.

Also, what do you mean by pooled data?

[luffy]

@grant.hovhannisyan, Thank you very much for the video tutorial, could you please share the code or link to the github code, it would be really helpful. About the pooled data, as i said in the above comments regarding the how the samples are pooled. What are your thoughts about it.

Thank you

[grant.hovhannisyan]

hi, drop me an email to [e-mail] and I will send you the materials