Questions & Answers

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  • Can this protocol use purified small RNA as starting material?

    Yes. Please note that we have seen high sample variability using some commercially available small RNA spin column purification methods, and validation of any purification method may be necessary. If purified small RNA is used as starting material, 10–50 ng should be used.

  • What is the minimum input material for small RNA sequencing?

    The protocol was optimized using 1.0 μg of total RNA.

Cluster Generation

  • Can pre-TruSeq libraries be run on TruSeq cluster kits?

    Yes, TruSeq cluster kits are backwards-compatible.


  • Why do I have reads that end in adapter sequences in my small RNA sequencing results?

    Reads longer than the small RNA molecule will sequence into the adapter. For this reason, we recommend sequencing for only enough cycles to cover the small RNAs of interest, or trimming the later cycles before aligning. The sequence that would need to be trimmed from the 3' end is given here for reference.


    This is the reverse complement of the index-specific primer sequence given in the Illumina Customer Sequence Letter, with the index sequence shown as nnnnnn.

  • Is a PhiX control lane necessary with small RNA libraries?

    Many small RNA libraries do not require a PhiX control lane to generate accurate matrix and phasing estimates. This is especially true in the case of samples from organisms with balanced genomes (e.g. mammalian organisms). Libraries from organisms with unbalanced genomes, or libraries with a high proportion of reads from a single template (for example, adapter reads, or samples from a tissue that expresses a single small RNA at high levels), may require use of a control lane for matrix and phasing generation. In any case, a PhiX control lane acts as a positive control for run performance and allows error rate estimation.

  • How many reads are needed per sample for small RNA sequencing?

    This depends on the application. For expression profiling, 1–2M mapped reads is a generally accepted range. For discovery applications, an increase to 2–5M reads may be considered.    

  • How many cycles of sequencing do small RNA libraries need?

    This depends on the aim of the experiment. For expression profiling projects, we do not recommend longer than 36 cycles of sequencing. The small RNA molecules are normally 15–30 bases long, and sequencing beyond this point only sequences the adapter. Eighteen cycles may be sufficient. For discovery projects, it may be worthwhile to do 40–50 cycles. This will sequence into the adapter, but the adapter sequence will be long enough to be unambiguously identified and removed even on longer small RNA molecules.


  • Are the Illumina adapter sequences available?

    Illumina adapter sequences are provided in the Illumina Customer Sequence Letter.

  • Can I compare data between the TruSeq small RNA protocol and earlier Illumina small RNA protocols?

    As with any molecular biology techniques, a change in protocol will give a change in absolute results. We do not recommend comparing absolute counts (or counts normalized to total counts) between different preparation protocols. However, comparison of fold change between the two protocols should yield good correlation.