RNASeq by Nextera vs. Standard

[eab]

Does anyone out there strongly prefer either Nextera or standard (TruSeq) library construction for RNAseq?

I use a version of the standard protocol, which is familiar to me, not fast but automatable, cheap-ish, and comes with a bit of insert-end bias that I understand is due to bias in random hexamer synthesis or priming.

My sense is that the Nextera protocol is faster, more expensive in consumables ($80/sample for Illumina kit), and generates a longer region of insert-end bias which theoretically causes greater skewing of expression data.

Agree?

Disagree?

Why?

[SS00]

Interested in this too. Currently testing protocols for Low Input RNA-Seq (500pg-2ng) and have been recommended the TotalScript Nextera by some colleagues.

Up until now I have been testing the SMARTer PolyA kit, but have been told Nextera TotalScipt allows Low Input Total RNA-Seq plus it's easier. I am not sure if the data will look as good as the SMARTer and honestly don't mind extra lab work if my sequencing data is better. Just looking at their protocol I see I have to decide between even coverage with 45% rRNA reads or low rRNA reads with 3' bias - not impressed!

Anyone have experience with the TotalScript kit or Nextera RNA-seq in general?

Sidenote: With such a low yield of RNA in a quiescent cell type (stem cells) I am worried doing Total RNA-seq will simply add to the ''noise'' in my analysis and make it harder to detect changes in lower-expressed RNA's. Any advice to this?

Sorry to hijack your thread eab!