Hello all and thanks in advance for any reply.
I want to perform RNA seq in human sperm samples and I want to have total RNA (mRNA + lncRNAs).
What are the parameters regarding read length, million reads etc that can offer the best quality results regarding sperm?
I know it is depleted of 18S/28S RNA and it is in general it has many small length fragments.. But I'd like to choose the best parameters for sequencing..
Hi Maria
There is no specific protocol for RNAseq for a group of cells. I understood from your question that you want to sequence the whole sperm, including cells. If this is the case and you want to analyze mRNAs+lncRNAs, and as a very basic rule of thumb, you would need to design the protocol to obtain a minimum coverage of around 50 million reads per sample for human cells (in pair ended mode). Of course if you can get more reads it would be better, because you will catch up the low-expressed lncRNAs.
If you contact a good service for RNAseq they will help you with the library preparation and ribodepletion, which actually I do not recommend to do by yourself unless you are a super-experienced technician in the field.
Read lenght that you can use is very variable, but in my hands between 50 and 150 should be OK for mRNAs+lncRNAs.
Best of luck
Paco
Thanks a lot for your answer Francisco.
It my first time utilizing RNA seq and i want to make sure it is done correctly
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA