TruSeq Small RNA Library Prep Kit FAQs

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  • Input


  • Yes. However, 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.

    • ChIP: 5–10 ng ChIP-enriched, fragmented DNA
    • DNA: 1 μg gDNA
    • DNA PCR-Free:
      • 1 µg gDNA for a 350 bp insert size
      • 2 µg gDNA for a 550 bp insert size
    • Nano DNA:
      • 100 ng gDNA for a 350 bp insert size
      • 200 ng gDNA for a 550 bp insert size
    • RNA: 0.1-1 μg total RNA
    • Small RNA: 1 μg total RNA
    • Targeted RNA Expression:
      • 50 ng total RNA
      • 100-200 ng degraded or FFPE RNA, depending on fragment size
  • Sequencing


  • RNA libraries should routinely be run as single-read (with an index read as desired). TruSeq libraries can be run as paired-end if desired (for example, for directional RNA custom preparations), but this is expected to offer no advantage and significant expense 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.

    No, not currently. The Directional mRNA-Seq Sample Preparation protocol would need to be optimized to take the longer adapters into account, as the size difference makes adapter dimers more difficult to distinguish from actual inserts using SPRI or column purifications. Illumina encourages customers to continue to use the Illumina Small RNA v1.5 adapters as specified in the Directional mRNA-Seq Sample Preparation Guide.

    This depends on the application. For expression profiling, 1–2M mapped reads is a generally accepted range. For discovery applications, an increase to 10–20M reads may be considered.