Total RNA-seq miRNA detection query

Question

[rob234king]

I have total RNA-seq sequencing (ribosomal RNA depleted) of single end 49bp stranded RNA from mouse. From total RNA there is these subclasses: mRNA, polyA RNA, polysomal RNA, tRNA, ribosomal RNA (removed), lincRNA, miRNA, piRNA, siRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, aRNA, crRNA, tasiRNA, rasiRNA, 7SK RNA.

My question is given miRNA is 20-24 nt and I have 49 bp RNA sequencing, I shouldn't have miRNA in my sequencing data as the size (nt length) is too small to be captured? Isn't a separate sequencing protocol needed if want the miRNA data from such an experiment?

I have gone down the route of: hisat2 -> featurecounts -> DESeq2 to perform differential expression using this data set. Anyone see a problem with the workflow that I used given mouse has a good genome and annotation to use.

Answer 1

[mark.ziemann]

Ribosomal RNA depleted RNA-seq won't capture small RNAs like microRNAs due to size. In RNA-seq, the RNA undergoes random priming for cDNA synthesis and there is a size selection step that excludes small fragments. To sequence small RNAs, a completely different library technique is used, which utilises an RNA ligase to stick on adaptor sequence to the insert smRNAs to allow them to be sequenced. That said, you might be able to detect low levels of primary miRNA gene transcript (before the hairpin gets cleaved) in rRNA depleted RNA-seq.

Your analysis pipeline seems reasonable.