Illumina Stranded mRNA Prep Ligation Protocol FAQs

Can any thermal cycler be used?

Illumina recommends one of the following thermal cyclers:

• Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad, catalog # 1851196)

• T100 Thermal Cycler (Bio-Rad, catalog # 1861096EDU)

Other thermal cyclers have not been validated by Illumina, but may be compatible; users must validate alternative thermal cyclers in their own labs. Alternative thermal cyclers must have a heated lid temperature setting as some steps require the lid to be set at 40ºC. Ensure that the thermal cycler meets this requirement.

Are there any intermediate QC steps?

No, there are no intermediate QC steps.

How is strandedness retained?

See Which reads map to each strand in Stranded RNA workflows, and how is strandedness achieved?

How to choose optimal index combinations?

For optimal index balancing see 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 or the TapeStation System and D1000 ScreenTape.

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

Illumina has validated storing dsDNA libraries at -20ºC for up to 30 days. It is recommended to perform library QC and quantitation immediately prior to sequencing.

What is the expected final library size?

The average fragment length is ~300-400 bp. The expected insert size is ~160 bp.

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

This could indicate the presence of bubble products; see Bubble products in sequencing libraries: causes, identification, and workflow recommendations for more information.

For any feedback or questions regarding this article (Illumina Knowledge Article #3290), contact Illumina Technical Support [e-mail].