> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/rna-enrichment-based-library-prep/library-preparation-rna-enrichment-based-library-prep-faq-list/000003294.md).
# Illumina RNA Prep with Enrichment, (L) Tagmentation Protocol FAQs
**What is eBLTL?**
eBLTL is the acronym for enrichment 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 recommends to use T100 Thermal Cycler, Bio-Rad part # 1861096. Thermal cycler with following specifications can also be considered.
* Compatible with semi-skirted 96-well PCR plates
* Heated lid
* Average ramp rate: 2°C/sec to -2.5°C/sec
* Maximum ramp rate: 2.5°C to 4°C
* Maximum reaction volume: 100 µl
* Minimum temperature control range: 4°C to 100°C
* Minimum temperature accuracy: ±0.5°C
* Minimum temperature uniformity: ±0.5°C
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](https://support.illumina.com/downloads/index-adapters-pooling-guide-1000000041074.html).
**Are there any intermediate QC points?**
Yes, at Normalize library step.
Qubit quantification using Qubit dsDNA BR Assay Kit.
\[Optional] Analyze 1 µl library with the one of the following analysis systems:
* Agilent 2100 Bioanalyzer System and DNA 1000 Kit.
* Agilent TapeStation System and D1000 ScreenTape.
**Which 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?**
No, 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 Qubit fluorometric method and the quality checked using a BioAnalyzer or TapeStation.
**What is the expected final library size?**
Typical libraries show a broad size distribution of 250-1000 bp.
**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, if libraries are stored.
**What is the shoulder of higher molecular weight that occasionally appears in library traces?**
This may be bubble product; see [Bubble products in sequencing libraries: causes, identification, and workflow recommendations](/library-preparation/general-library-prep/library-preparation-general-library-prep-reference_material-list/000001918.md) for additional information.
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3294), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003294%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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