I'm studying "Deep sequencing the circadian and diurnal transcriptome of Drosophila brain" Hughes et al., 2012. I've got some problems with the materials and methods.
Before RNAseq, the authors amplify RNA. They use two kits: Poly(A) based and non-poly(A) based. For the samples Poly(A) based amplified they do a sequencing to generate 100 bp paired-end and for the samples Non poly(A) based they do a sequencing to generate 75 single-ends.
I read in the strategy:
Total RNA was amplified, and ribosomal RNA was depleted, using a non-poly(A)-based amplification kit, which significantly diminishes the 3' bias in downstream libraries used for RNA-seq (see Methods).
1) I don't understand why they use both kits. Actually, they say that they want to asses the bias introduced by an alternative amplification methods BUT the following sequencing is not the same in both cases so there is more than one parameter which change between the both protocols so I don't understand how they can compare.
2) For the poly(A) based I understand that the rRNA are not polyadenylated so they remove rRNA from the amplified sample. However, don't they remove non-coding RNA too (long or short)?
3) For the non poly(A) based, I don't understand how they remove the rRNA but I understand that since they don't select the RNA according to their poly(A) tails they keep the rest of the RNAs which are not kept in the poly(A) based amplification.
4) When I look to the RUM statistics, I see that there is less total aligned read in non-poly(A) based: 70-80% (non polyA) and 80-90% (polyA). So it seems like we loose some information with non-poly(A) based.
To resume, why the authors use Poly(A) and non-poly(A)? Why do they show all the rest of the results from the non-poly(A) amplification since they have less aligned read? How do they compare since the following sequencing is not the same? How the rRNA are removed in both techniques and especially in non-poly(A)?
I didn't find the protocols kit with exactly the same name but here are the links to the protocols which are closer (I think).