TruSeq RNA Sample Prep Kit v2 FAQs

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  • Input


  • Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than or equal to eight.

    • 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


  • For runs on HiSeq Systems, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV.

    If you are setting up a run in MiSeq Control Software or analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. 

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

    • TruSeq DNA and RNA Sample Prep v1 kits support 12-plex per lane and 96-plex per flow cell.
    • TruSeq DNA v2/LT and RNA Sample Prep v2 kits support 24-plex per lane and 192-plex per flow cell.

    For TruSeq v2/LT kits, standard sequencing primers included in the cluster generation kits are required.

    For TruSeq HT kits, primer usage will depend on the flow cell type used. Primers in the TruSeq Dual Index Sequencing primer kit, single read (catalog # FC-121-1003) are needed to run TruSeq HT Dual-Indexed libraries on single read v3 flow cells.  These primers are HP10 (read 1), HP12 (Index read 1), HP9 (Index read 2 for single read flow cells only).  This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on single-read rapid flow cells, or paired-end v3 or Rapid flow cells for HiSeq, HiScanSQ, GAIIx. The primers in the TruSeq Dual Index Sequencing Primer kits are backwards compatible with all Illumina libraries; there is no need to spike in primers as these primers are backwards compatible with other Illumina library types.