Protocol FAQs for Illumina Stranded Total RNA Prep Ligation with Ribo Zero Plus

1 Can any thermal cycler be used?

The thermal cycler must have a heated lid temperature setting as some steps require the lid to be set at 40°C or 70°C. Furthermore, one step requires a gradient of 0.1°C/second. Verify that the thermal cycler meets these requirements.

2 What are the safe stopping points?

There are three safe stopping points:

· after second-strand cDNA synthesis

· after the clean-up step that follows ligation

· after the amplification step.

3 Are there any intermediate QC points?

No, as the amount of RNA is limited.

4 How is stranded cDNA generated?

Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Master Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. This specificity is accompanied by the addition of Actinomycin D to the First Strand Master Mix Act D (FSA), which prevents spurious DNA dependent synthesis during first strand synthesis, allowing RNA-only dependent synthesis.

H How is strandedness maintained after DNA amplification?

Stranded information is maintained via the directionality of the ligated adapters.

H How do to choose optimal index combinations?

For optimal index balancing refer to the Index Adapters Pooling Guide.

What is the recommended final library QC method?

Libraries can be checked using the Agilent 2100 Bioanalyzer and DNA 1000 Kit. It is also possible to do a further quantification check with the Qubit dsDNA BR Assay Kit.

How to store final libraries and for how long are they stable?

Illumina has tested storing double stranded DNA libraries at -20ºC for up to 7 days. It is also recommended to perform library QC and quantitation immediately prior to sequencing, following storage.

9 What is the expected final library size?

The average fragment length is ~350-400 bp. The expected insert size is ~200-250 bp.

What is the shoulder of higher molecular weight that occasionally appears in library traces?

This could be bubble product. See the following for more information: Bubble products in sequencing libraries: causes, identification, and workflow recommendations

How long does it take to generate ready-to-sequence libraries?

Typically, ≤8 hours total turnaround time.

For any feedback or questions regarding this article (Illumina Knowledge Article #3286), contact Illumina Technical Support [e-mail].