What are the main differences between TruSeq Stranded and Illumina Stranded kits?
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Labs choosing an RNA-Seq kit may want to understand the differences between the newer Illumina Stranded kits and the previous TruSeq Stranded RNA-Seq kits. This article is meant to provide additional information to supplement the following article.
Below are some of the main differences between the kits.
Input amount
TruSeq Stranded Total RNA/mRNA kits are compatible with 100-1000 ng input.
Illumina Stranded Total RNA kit is compatible with 1-1000 high-quality (RIN ≥ 9) RNA or 10-100 ng low-quality (RIN ≥ 2 or FFPE DV200 >55) input.
Illumina Stranded mRNA kit is compatible with 25-1000 ng high-quality input.
Total assay time
Illumina Stranded Total RNA workflow time is ~7 hours.
Illumina Stranded mRNA workflow time is ~6.5 hours.
TruSeq Stranded mRNA/Total RNA workflow time is ~11.5 hours.
Index options
Illumina Stranded RNA kits are used with the IDT for Illumina RNA UD Index kits and Illumina RNA UD index kits. Up to 384 samples can be multiplexed with Sets A-D. For TruSeq Stranded RNA kits, the highest multiplexing capacity is 96 with the TruSeq RNA UD Indexes.
Illumina Stranded RNA kits use a ligated anchor which makes the libraries compatible with IDT for Illumina RNA UD Indexes and Illumina RNA UD indexes; TruSeq Stranded does not use an anchor ligation step, rather the full adapters are ligated in a single step.
Workflow and reagents
Illumina Stranded RNA workflows and reagents are optimized to be more efficient, and compatible with lower input.
Illumina Stranded RNA kits include the RVT reverse transcriptase reagent; whereas, in TruSeq Stranded RNA kits, the reverse transcriptase must be purchased separately from a third-party vendor.
In Illumina Stranded RNA protocols, there are a variable number of PCR cycles based on the input quantity and quality. TruSeq Stranded RNA workflows have a set number of PCR cycles.
Illumina Stranded RNA libraries have slightly larger final library size (average size 375-475 bp for Total RNA, 300-400 bp for mRNA) as compared to TruSeq Stranded RNA libraries (average size 260 bp).
When using FFPE samples with TruSeq Stranded Total RNA, the fragmentation time may need to be modified. There is no fragmentation modification time when using FFPE samples with Illumina Stranded Total RNA.
Ribo-Zero options
Illumina Stranded Total RNA is bundled with Ribo-Zero Plus, which uses enzymatic rRNA depletion rather than bead-based depletion method. Ribo-Zero Plus targets more rRNA species.
TruSeq Stranded Total RNA can be used with standalone Ribo-Zero Plus, but it is not bundled with the library prep reagents. TruSeq Ribo-Zero Gold, Human/Mouse/Rat, Plant, and Globin all use a magnetic bead-based pulldown for rRNA depletion.
Kit size
Illumina Stranded RNA kits are available in 16-sample and 96-sample configurations.
TruSeq Stranded RNA kits are available in 48-sample and 96-sample configurations.
For any feedback or questions regarding this article (Illumina Knowledge Article #5928), contact Illumina Technical Support .