2D Complete RNA

FAQs

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Does this prep retain strand information?

Yes, its retains >99% strand accuracy.

Library Molecule Structure

What are the sequencing recommendation?

We recommend Sequencing with Single-end reads. This can save on sequencing cost.
  • The first 8 bases on R2 are a random tag sequence which can be used as an UMI.
  • For Pair-end reads, there can be a significant drop in read quality for R2 because of the presence of Poly T sequence.
  • With single-reads, using 2D Complete RNA kit, you can get coverage of full transcript as shown with all the data on this page.

Can we do pair-end reads?

We don't recommend.

Example BioAnalyzer Traces

What are the recommendation for Fragmentation?

Optimum fragmentation depends on several factors includingRNA quality. We recommend user to optimize a Fragementation protocol that meets your requirement. In the below image, we used Universal Human Reference RNA as sample and did a time course to show the fragmentation profile. This might help you to get going.

Does the kit include sample indices?

Yes it does. For kit size of 6 reactions, 6 dual index Sample Index (SImix) is included. For kit size of 24 reactions, 24 dual index Sample Index (SImix) is included. SI-mixes are in separate box than the Library Prep kit components. For sequence information of the sample indices, please check the SAMPLE INDICES button right next to the FAQs button on the Product ordering page.

Can I use my own sample indices?

We strongly recommend using the provided Sample indices as they are optimized to reduce artifacts. However, if you do wish to use your own sample indices, please contact us and we can help with the design of the oligo sequences.

Are 2D Library Prep kits compatible with Ion Torrent Platform?

No. At the moment, all 2D kits are designed and optimized for Illumina Platforms.

Does the DNA need to be removed from the sample prior to Library Prep?

Yes. We recommend strand protocols to remove DNA from the sample prior to using it with 2D Complete RNA Kit.

Can I use this kit with FFPE sample or Low Quality RNA?

Yes. The 2D Complete kit can accommodate broad range of input amounts and RNA quality range.

Are all 2D RNA Library Prep Stranded?

Yes. We do not have a non-stranded version of any kits.

Does the resulting Library has UMI?

The first 8 bases on R2 are a random tag sequence which can be used as an UMI. With the 2D Complete RNA kit, we recommend to only acquire 8bp of R2 and have longer R1.

Do I need to Ribo Deplete?

Yes. For best utilization of Sequencing Run, we recommend to either ribo deplete or enrich for mRNA using any standard commercial kit or 2D provided solution.

What is the difference between your 2D Complete RNA and 2D Total RNA kit?

2D Complete Kit has extremely high sensitivity and is designed to capture all RNAs ranging from miRNA to Long RNA. You can use as little as 100pg input for the library prep. 2D Total RNA Kit has a very simple workflow, and you can do standard pair-end reads since there is no PolyA tailing step with this method. The minimum required amount of sample for 2D Total RNA kit is 10ng.

Why do we add Poly A tails?

This is to significantly boost the conversion efficiency and truly capture ALL RNA biotypes in the single library Prep. The ligation based approaches are best suited for small RNA, since long RNA has high secondary structures. The random priming approaches are biased for Long RNA and has very limited capability to capture short RNA and completely not suited for miRNA. the PolyA tailing approach allows us to capture ALL RNAs without compromise.

Do I have an option to not add PolyA tail?

We really think you should give the 2D Complete RNA kit a go, we feel confident that the data will impress you. If you do not want to add PolyA tail, you can switch to 2D Total RNA kit, which does not require PolyA tailing.

Which RNA isolation method/Kit should I use?

Various RNA isolation methods differ significantly particularly in recovery small RNA, please check the References.
  • Quantification of SmallNon-Coding RNAs Allowsan Accurate Comparison of miRNA Expression Profiles. AndreaMasotti, VivianaCaputo, LetiziaDaSacco, AntonioPizzuti, Bruno Dallapiccola, and Gian Franco Bottazzo. Journal of Biomedicine and Biotechnology Volume 2009, Article ID 659028.
  • A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. Marlo K Sellin Jeffries, Andor J Kiss, Austin W Smith and James T Oris. BMC Biotechnology, 201414:94

How deep do I need to sequence my library?

It depends on your specific sample type and application. Here is some data that might provide you with general guidlines of sequencing depth vs library complexity using 2D Complete RNA kit. This data was generated using 2D Complete RNA kit with Universal Human Reference RNA (10ng) as template, and sequenced on Illumina HiSeq 2500 (1x100bp). Data analysis was done on Illumina BaseSpace RNA Alignment Application.

How many PCR cycles I need to do for Library Amplification?

Can I place an order using a PO number?

Yes, you can! For fast service, we do encourage you to use our web ordering. However, you can place an order using a PO #. Orders placed using PO might take an extra day for processing.

If you're placing the order for the first time using a PO number, email us at info@2dgenomics.com to setup your account.

For customers with existing accounts, please click here - PO Order Form