Illumina RNA Prep Tagmentation (L) with Enrichment Protocol FAQs

What is eBLTL?

eBLTL is the acronym for nrichment Bead-Linked Transposomes - L for the Illumina RNA Prep Tagmentation (L) with Enrichment workflow. eBLTL are magnetic beads with immobilized Tagmentation Enzymes.

How should eBLTL be stored?

eBLTL must be stored at 2-8ºC in an upright position to make sure beads are submerged in solution.

Can eBLT from the Illumina DNA Prep with Enrichment kit or BLT from DNA Prep kit be used interchangeably?

No, other bead-linked transposomes have different formulations.

Can any thermal cycler be used?

Illumina has validated the protocol using the Bio-Rad C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module. Labs may decide to validate the protocol with another thermal cycler. Some steps require the lid temperature to be at 40ºC or 70ºC so the cycler should meet this requirement. Labs will need to test to make sure any thermal cycler produces the expected results.

How to choose optimal index combinations?

For optimal index balancing, refer to the index adapter pooling guide.

What are the safe stopping points?

There are 6 safe stopping points:

• after the cDNA clean-up step

• after PCR amplification of tagmented DNA

• after clean-up of the pre-enriched library

• after the elution following the 3 wash steps

• after the enrichment PCR

• after clean-up of the final library

Are there any intermediate QC points?

Yes, Qubit quantification only.

What quantification method is recommended before starting enrichment?

The Qubit dsDNA BR Assay Kit.

What is the recommended amount of library for enrichment?

Use 200 ng of each library. Uneven library input amount into enrichment is not expected to change enrichment efficiency. However, it may impact the per sample coverage in the sequencing data.

Can more than 3-plex be used for the enrichment?

This is not recommended.

Can libraries be pooled by volume instead of mass for enrichment?

Pooling libraries by volume can be considered; however, for the most consistent results, Illumina recommends pooling by mass.

What is the recommended final library QC method?

Final library must be quantified using a fluorometric method and the quality checked using a BioAnalyzer.

What is the expected final library size?

The average fragment length is ~380-450 bp. The expected insert size is ~200-250 bp.

How to store my 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, if libraries are stored.

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

This may be bubble product.

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

Typically, <9 hours total turnaround time for the library prep and enrichment workflow.

For any feedback or questions regarding this article (Illumina Knowledge Article #3294), contact Illumina Technical Support [e-mail].