TruSeq Stranded Total RNA FAQs

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  • General

  • The TruSeq Stranded Total RNA library preparation kit constructs stranded total RNA libraries that are compatible with Illumina sequencing platforms for Next Generation Sequencing. The workflow is based on rRNA depletion and targets long RNA species, which are primarily mRNA and long, non-coding RNA, for library preparation. This workflow requires as little as 100 ng of input RNA and is compatible with degraded, FFPE input RNA. 

    TruSeq Stranded mRNA specifically targets poly-adenylated RNA (including coding RNA and non-coding with poly adenylation), while TruSeq Stranded Total RNA prepares RNA libraries based on rRNA depletion, targeting coding RNA and long non-coding RNA.

    TruSeq Stranded mRNA requires high quality RNA (RIN>8) as input and TruSeq Stranded Total RNA can work with degraded / FFPE samples.

    The following library prep and index adapter components are available to order through Illumina. From Illumina, order one catalog number for the library prep component and one catalog number for the index adapter component depending on the number of samples for your experiment.

    Library Prep Component

    Catalog #

    TruSeq Stranded Total RNA (48 samples)- Human/Mouse/Rat


    TruSeq Stranded Total RNA (96 samples)- Human/Mouse/Rat


    TruSeq Stranded Total RNA (48 samples)- Human/Mouse/Rat- Gold


    TruSeq Stranded Total RNA (96 samples)- Human/Mouse/Rat- Gold


    TruSeq Stranded Total RNA (48 samples)- Plant


    TruSeq Stranded Total RNA (96 samples)- Plant


    TruSeq Stranded Total RNA (48 samples)- Globin


    TruSeq Stranded Total RNA (96 samples)- Globin




    Index Adapter Option


    IDT for Illumina- TruSeq RNA UD Indexes (24 indexes)


    IDT for Illumina- TruSeq RNA UD Indexes (96 indexes)


    TruSeq RNA CD Indexes- 96 Indexes


    TruSeq RNA Single Index Set A- 12 Indexes


    TruSeq RNA Single Index Set B- 12 Indexes


    Single index adapters are provided in tubes.  Each i7 indexed adapter is paired in its tube with the same universal adapter that contains no (i5) barcode.

    Combinatorial Dual Indexed Adapters are plated pairs of adapters such that every i5 indexed adapter would be paired with each i7 indexed adapter in a arrayed matrix across the plate.

    Unique Dual Indexed Adapters are plated pairs of adapters such that every i5 indexed adapter and every i7 indexed adapter is only used once on a plate. With unique dual index adapters, identification and filtering of indexed hopped reads allow customers to confidently multiplex their samples.

    Illumina recommends using Unique Dual Indexed Adapters for library prep and sequencing on Illumina sequencers.

    The choice of TruSeq Stranded Total RNA kit depends on the input.

    • For human, mouse, or rat samples, choose TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat or Ribo-Zero Human/Mouse/Rat Gold.
    • For human, mouse or, rat blood samples, use TruSeq Stranded Total RNA with Ribo-Zero Globin, which depletes both rRNAs and globin mRNAs.

    The main difference between the two options is that Ribo-Zero Human/Mouse/Rat Gold also depletes mitochondrial rRNAs.

    The Ribo-Zero Globin removal mix targets cytoplasmic rRNA, mitochondrial rRNA, plus globin mRNA.

    The Ribo-Zero Plant removal mix targets cytoplasmic (25S, 18S, 5.8S, and 5S rRNA), Chloroplast (23 S, 16S, 5S, and 4.5S), and mitochondrial rRNA (18S and 5S).

    The Ribo-Zero Globin removal mix targets cytoplasmic rRNA, mitochondrial rRNA, plus globin mRNA.

    The Ribo-Zero Globin kit is tested to deplete Globin mRNAs from human, mouse and rat blood samples. Use of this kits with other organisms has not been tested and is not supported.

    The Ribo-Zero Globin kit has been specifically tailored for blood samples from human, mouse, or rat. However, TruSeq Stranded Total RNA with Ribo-Zero Globin will deplete rRNA and can be used with samples other than blood.

    The Ribo-Zero Plant kit contains oligos that will remove cytoplasmic and chloroplast rRNA from leaves, seeds and roots.

    No, the recommended SuperScript II and Agencourt AMPure XP 60 ml Kit are user supplied.

    Yes. This will allow to multiplex up to 24 samples if the samples can be covered at the expected coverage. See “What is the recommended read length and type” in the Sequencing section.

  • Input

  • TruSeq Stranded Total RNA can work with high quality as well as partially degraded total RNA. When working with partially degraded RNA, please refer to the Appendix B “Alternate Fragmentation Protocol” and “Modify RNA Fragmentation Time for Degraded RNA” for recommended modified fragmentation time.

    For degraded RNA from human samples, TruSeq RNA Access can be also considered as an alternative, which specifically targets coding RNA for library preparation. 

    For degraded RNA from species other than human, mouse, rat or plant, consider standalone Ribo-Zero reagents and prepare the RNA libraries with TruSeq Stranded mRNA sample preparation kit. Modification of fragmentation time will need to be taken into considerations; please contact Illumina Technical Support for further suggestions. 

    The optimal 260/280 and 260/230 ratios for a clean RNA sample are expected to be ~ 2.0 and ≥ 2.0.

    0.1–4 μg total RNA is supported and expected to yield libraries of similar qualities. For highly degraded RNA, using input amount on the higher end of the 0.1-1 ug range can be considered. 

    A fluorometric method such as Qubit or Ribogreen is recommended. A measurement based on UV/VIS spectrophotometry scanning such as NanoDrop, will quantify all nucleic acids, which can result in overestimation of input material and impact library quality.

    DNase treatment is recommended. This will ensure accurate quantitation of the input material and prevent any possible interference of contaminating gDNA in library preparation. 

    TruSeq Stranded Total RNA will mainly target mRNA and long non-coding RNAs.

    This protocol will not capture small RNAs. If we are interested in sequencing small RNAs, we can consider TruSeq Small RNA library preparation kit. 

    Illumina recommends using Agilent Technologies Human UHR total RNA (catalog # 740000) as a positive control sample.

  • Protocol

  • Inline controls are nucleic acid fragments added to the library prep sample as internal control to examine the success of a particular step during library preparation process. This can be helpful in troubleshooting the library preparation process when analyzing the sequencing data and contribute to about 0.1% of the total sequencing data.

    The use inline controls are optional; if inline controls are not being used, RSB buffer can be added.

    Illumina has optimized the workflow to increase ease-of-use and scalability. Changes include the use of plates compatible with multi-channel pipettes, the streamlining of incubation steps, and an overall reduction in hands-on time.

    For standard (non-FFPE) samples, less than 6% of ribosomal RNA contamination is expected. For FFPE samples, less than 6% is expected. These values vary depending on tissue quality and source.

    It is possible to use larger fragment sizes, however, we have found that shorter fragment sizes generate the best coverage. Refer to Appendix A in the referencen guide for more information.

    The safe stopping points in the protocol include:

    • Post second strand cDNA synthesis and cleanup
    • Post adapter ligation and cleanup
    • Post PCR amplification and cleanup

    These tested safe stopping points are labeled in the reference guide.

    We have not tested intermediate QC points as the intermediate products may be limited. 

    Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide.

    This specificity is accompanied by the addition of Actinomycin D to the First Strand Master Mix Act D (FSA), which prevents spurious DNA dependent synthesis during first strand synthesis, allowing RNA-only dependent synthesis.

    When sequencing a limited number of samples, it is important to maintain color balance for each cycle of the index read being sequenced to prevent registration failure for index read sequencing.

    For sequencing platforms that utilize 4-color chemistry such as Hiseq 2500, G/T bases are sequenced with a green laser and A/C bases with a red laser. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. Please refer to the Index Adapter Pooling Guide for recommendations for pooling limited TruSeq Stranded mRNA samples.

    For sequencing platforms that utilize 2-color chemistry such as Nextseq 500/550, two fluorescent dyes are used for sequencing: Red for C, Green for T, Green and Red for A and no dye for G. To ensure proper index registration, best practices are to use indexes that begin with at least 1 base other than G in either of the first 2 cycles and provide signal in at least 1 channel, preferably both channels, for every cycle. See bulletin “Library pooling guidelines for the Nextseq and MiniSeq systems” for more information.

    Longer drying times may be required depending on environmental variability and the amount of ethanol remaining in the well.  

    Use qPCR to quantitate the libraries. Samples should be quantified prior to pooling to achieve even pooling.

    When preparing the libraries, it is important to choose indexes unique for each sample that will be multiplexed together and that this group of indexes is color balanced.

    Color balance during the index read is necessary to ensure proper image registration. To ensure color balancing, make sure there is a mixture of each of the color channels (red or green) at each cycle so that the image is properly registered and a base call will be generated with high quality. If no base is called, the index read may not be able to be matched with the sequence specified in the sample sheet, resulting in demultiplexing failure.

    Refer to the following two questions “How do I choose the best index combinations for my sample prep” and “How do I choose index combinations if I pool a smaller number of samples (<12)” for more information.

    Illumina’s proprietary method ensures ligation of 2 different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. 

    We have tested storing dsDNA libraries at -20ºC up to 7 days. For longer storage, Illumina recommends adding Tween-20 to a final concentration of 0.1% and storing concentrated libraries in a LoBind tube. It is also recommended to perform library QC and quantitation immediately prior to sequencing.

    Peaks around 120-130 bp may represent adapter dimers. These can be removed with an additional bead cleanup step.

  • Sequencing

  • Download Illumina Experiment Manger and choose the index adapter option you have chosen for your experiment when setting up the Sample Sheet.

    If we are sequencing on the Nextseq System, we can set up indexing information in PrepTab on BaseSpace or BaseSpace OnSite Sequence Hub to demultiplex on BaseSpace.

    After DNA amplification, stranded information is maintained via the directionality of the ligated adapters. Briefly, the orientation of the adapters in the libraries and the sequencing direction on the Illumina platform determine that Read 1 sequences will be identical to the antisense strand of DNA, which is also the first strand cDNA. 

    Only the first strand cDNA is amplified and sequenced. 

    For runs on the HiSeq 2500 and HiSeq 3000/4000, sample sheets are optional unless BaseSpace Sequence Hub or BaseSpace Onsite Sequence Hub will be used to perform data analysis. In addition, using a sample sheet for a HiSeq 2500 run allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run.

    NextSeq and MiniSeq runs do not require a sample sheet to start the run either. If the data are being streamed to BaseSpace Sequence Hub or using BaseSpace Onsite Sequence Hub, setting up the run Prep tab will allow FASTQ generation and visualization of indexing data on the Indexing QC tab on BaseSpace.

    For runs on any sequencing instrument, if demultiplexing will be performed with bcl2fastq2 conversion software, a sample sheet is required.

    MiSeq runs on the other hand, require a sample sheet when setting up the run.

    Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) software prior to performing library prep in order to confirm appropriate index combinations.

    The read length and format (single read versus paired end) are important considerations in the design of RNA sequencing experiments. The table below provides guidance on some factors to consider. These recommendations are based on internal and external data, but do not represent strict cut-offs. Needs for individual projects may vary based on multiple variables as well as user preference. Literature needs to be consulted for the most up to date resources.


    Read Type

    Read Depth/sample (mRNA/Total RNA) (Millions of Reads)

    Gene profiling (gene-level counts)

    1 x 50

    >5 M / >10 M

    Discovery (alternative transcripts, gene fusions, etc.)

    2 x 75

    ≥50 M / >100 M

    Complete transcriptome annotation

    2 x 75 - 100

    ≥100 M / ≥200 M

    Due to the directional nature of the TruSeq Stranded RNA assays, paired-end sequencing captures both ends of the RNA fragment. Therefore, paired-end sequencing can help with data alignment, discovery of novel transcripts, and identification of PCR duplicates.

  • Analysis

  • Use the RNA-seq Alignmentand Cufflinks Assembly & DE apps on BaseSpace Sequence Hub to analyze data. 

    The RNA-seq Alignment app will perform read alignment, variant calling, read counting and detect RNA fusions. The Cufflinks Assembly & DE app on the other hand, will perform novel transcript assembly, differential expression analysis and FPKM abundance estimates.

    On instrument analyses are currently not supported. 

    The RNA-seq Alignment app reports the % Stranded reads, which is a calculation of the percent aligned Read 1 reads that are mapped to the antisense gDNA strand, and the percent aligned Read 2 reads that are mapped to the sense gDNA strand.