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Goal/Experiment:
The goal of the experiment is to assess the impact of technical bias and sample complexity in RNA sequencing by using RNA sequins as internal controls. RNA sequins act as synthetic genes to normalize and control variations in RNA sequencing experiments.

# Addition of RNA Sequins to Samples for RNA Sequencing

**Version 1**  
**Tim Mercer**  
**Garvan Institute of Medical Research**  
[doi.org/10.17504/protocols.io.x8cfrsw](dx.doi.org/10.17504/protocols.io.x8cfrsw)

## Abstract
RNA sequencing can measure both gene or isoform expression and reconstruct novel and complex spliced isoforms. However, the sheer size and complexity of the transcriptome, as well as technical bias, can confound analysis with RNA-seq. To assess the impact of these variables, we developed a set of RNA sequins that represent synthetic genes that act as internal controls during RNA sequencing.

Each RNA sequin represents an individual isoform, with multiple isoforms forming artificial gene loci that are encoded within the *in silico* chromosome (chrIS). By modulating the relative abundance of individual sequin isoforms, we can emulate alternative splicing while modulating the abundance of multiple isoforms to emulate gene expression. Accordingly, RNA sequins are mixed at different concentrations to emulate differences in gene expression and alternative splicing.

By sequentially diluting sequins, we can establish a reference ladder across a range of gene expressions. We formulate multiple alternative mixtures that differ in the concentration of individual sequins. By comparing mixtures, we can emulate differential gene expression and alternative splicing between samples. By contrast, RNA sequins with invariant concentrations between mixtures provide static scaling factors that enable quantitative normalization between multiple RNAseq libraries.

The RNA sequin mixture is added to a user’s RNA sample at a fractional concentration prior to library preparation. The combined sample and sequins then undergo sequencing. The sequins can then be distinguished in the output library by their synthetic sequence, and analyzed as internal controls.

For further detailed background on the design, validation, and use of sequins, we refer users to "Spliced synthetic genes as internal controls in RNA sequencing experiments" by Hardwick et al., (2016) Nature Methods.

**External Link:**  
[www.sequinstandards.com](www.sequinstandards.com)

**This protocol accompanies the following publication:**  
**Hardwick et. al., Spliced synthetic genes as internal controls in RNA sequencing experiments. (2016) Nature Methods.**

## Protocol Status
**Working**  
We use this protocol in our group, and it is working.

## Materials

| Name                | Catalog # | Vendor  |
|---------------------|-----------|---------|
| RNA sequins standards | [View](https://www.sequinstandards.com)   | Sequins |

## Step Materials

| Name                | Catalog # | Vendor  |
|---------------------|-----------|---------|
| RNA sequins standards | [View](https://www.sequinstandards.com)   | Sequins |

### Re-suspension and Storage of Sequins

1. **RNA Sequins Standards**  
   ![RNA Sequins Standards](https://www.sequinstandards.com)

   Upon receipt of RNA sequins, first check to ensure they have not thawed during shipment and immediately transfer the RNA sequins to frozen storage at -80°C (sequins should not be stored in a -20°C frost-free freezer).

   ![RNA Sequin Mixture Traces](https://www.sequinstandards.com)

   *Figure 1. Example traces of RNA sequins using an Agilent 2100 BioAnalyzer with the RNA Nano Kit (Agilent Technologies) for (left upper) neat Sequin Mixture A and (left lower) neat Sequins Mixture B. Also shown are example traces for (right upper) K562 with Sequin Mixture A and (right lower) GM12878 with Sequins Mixture B.*

2. Each tube contains RNA sequins provided in solution in 10 µL nuclease-free water at a concentration of 15 ng/µL. On first thaw, spin the tube down to collect the contents at the bottom of the tube, and prepare smaller single-use aliquots to minimize subsequent freeze-thaw cycles. The exact amount of RNA sequins required for a single-use aliquot depends on the sample input required for your preferred library preparation method.

   **Table 1. Guidelines for diluting RNA sequins according to sample RNA amounts (recommended 1% spike-in).**

   | Sample RNA | Sequin Mass | Sequin Volume (dilution from 15 ng/µL stock)|
   |------------|-------------|---------------------------------------------|
   | 20 ng      | 0.2 ng      | 1 µL (1:75)                                 |
   | 50 ng      | 0.5 ng      |  1 µL (1:30)                                |
   | 100 ng     | 1.0 ng      |  1 µL (1:15)                                |
   | 500 ng     | 5.0 ng      |  1 µL (1:3)                                 |
   | 1000 ng    | 10.0 ng     |  1 µL (2:3)                                 |

#### Addition of Sequins to Sample, Library Preparation, and Sequencing

3. The diluted RNA sequins should then be added directly to the sample RNA prior to any subsequent processing steps (such as poly-A enrichment or rRNA depletion). While this enables an assessment of these processing steps, the amount and dilution of RNA sequins added may need to be modified accordingly.

   > **COMMENT:** RNA sequins are provided in two alternative mixture formulations: Mix A and B. Each contains the same sequin isoforms, however they have been formulated at molar ratios. This emulates fold-change differences in gene expression and alternative splicing between the two mixtures. If a gene-profiling RNAseq experiment is being performed to identify differences in gene expression and splicing between two conditions, it is suggested that Mixture A and B be added alternately to separate samples from each condition being compared (ensure that both mixtures are not added to a single sample). This enables the use of RNA sequins to assess the detection of fold-change differences between samples.

4. Use the combined sample and sequins as input according to the protocol of your preferred library preparation kit.

   > **COMMENT:** The downstream library preparation workflow may require the user to concentrate the sample RNA after the addition of the RNA sequins. RNA samples can be concentrated using either ethanol precipitation, SPRI® bead purification (e.g. RNAClean® XP, Beckman Coulter), column-based methods (e.g. RNA Clean & Concentrator™ Kit, Zymo Research), or using vacuum centrifugation.

   ![Successful Sequenced Libraries](https://www.sequinstandards.com)

   *Figure 2. Successful sequin-containing (total) RNA Libraries. A, K562 with Sequins Mix A. B, GM12878 with Sequins Mix B. Samples analyzed by Agilent 2100 BioAnalyzer trace (size distributions sequenced on an Illumina HiSeq 2500 Instrument).*

5. The library that is generated from the combined RNA sample and sequins is then sequenced per manufacturer’s instructions. In this example, Illumina HiSeq 2500 was used.

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