What is the number of reads would you recommend to analyze lncRNAs without loss of other transcripts such as miRNAs or mRNAs, but as cheaper as possible?

Question

[ismael.riquelme.contreras]

What is the number of reads recommended to analyze lncRNAs without loss of other transcripts such as miRNAs or mRNAs, but as cheaper as possible? I want to performed RNA-seq in tissue samples and cell lines, focusing mainly on lncRNAs patterns in different conditions. In addition, it would be very useful to use the same determination to study simultaneously the differential expression of mRNA and miRNAs. I know a depletion of ribosomal RNAs is needed. However, I do not know what number of reads (depht) can be used to define these transcripts in a cost-benefit meaning, because my money is scarce. I will be attentive to your comments. Thanks.

[i.sudbery]

Using any standard RNA-seq protocol you can't analyse miRNAs and mRNA/lncRNA in the library prep. Almost all library prep protcols involve a size selection. miRNAs are around 22nt and require specialist methods to prepare libraries from them. lncRNAs/mRNAs usually involve selecting fragment sizes in the 200-300nt range.

[GenoMax]

Here are some numbers to get you started.

[WouterDeCoster]

If you want to save money you could also have a look at Lexogen Quantseq for sequencing only a 3' fragment per mRNA molecule - and hence sequence less because you are not covering the gene body. But obviously, whether that approach makes sense depends on your biological question. Since this protocol uses polyA based capture there is no need for rRNA depletion.

Note that not all lncRNAs are poly-adenylated AFAIK, but many are.