Hello there,

Today I was QC-ing some RNAseq data. My basic experiment looks to compare the RNA profiles from a cell line at three levels: - Total RNA - Monosome-associated RNA - Polysome-associated RNA (the last two which were extracted following gradient fractionation (i.e. polysome profilling)

RNAseq library prep was oligoDT enrichment and DNAse treatment before multiplexing.

Following mapping I realised that monosome and (slightly lesser-degree) polysome-associated RNAs had lower exonic % mapping vs paired total RNA samples, slightly more mitochondrial and ribosomal RNA, and greater duplication.

I speculate that the monosome and polysome-associated RNAs by nature have lower diversity as we are only sequencing the actively translated mRNAs (which may explain the duplication % increase (i.e. en masse sequencing of select few transcripts) - but does anyone know why exon % mappings would decrease?

For reference - my total RNAs mapped to 60% exons, Polysome-RNAs mapped to 40-50% exons, and Monosome-RNAs mapped to 10-20% exons.

My monosome samples are not essential but the Polysome-RNAs are fundamental to my study - is a variable exonic representation between 40-50% useable for downstream analyses?

If anyone has any experience that could help me that would be great!