Use 10 ng of high-quality universal human reference total RNA as input. If starting with FFPE RNA, the sample input amount is based on sample quality. Use the percentage of RNA fragments > 200 nt fragment distribution value (DV200) as a reliable determinant of FFPE RNA quality.
Input Requirement Per Reaction
|Library Prep: 20-40 ng|
|Library Prep: 40-100 ng|
For more information, see the Evaluating RNA Quality from FFPE Samples tech note.
Use a fluorometric assay (ie, RiboGreen or Qubit) to accurately quantify total RNA input. For more information, see the reference guide.
Use the Fragment Analyzer to quantify RNA samples. For more information, see Evaluating RNA Quality from FFPE Samples.
Coding Exome Oligos (CEX)
There are eight safe stopping points in the TruSeq RNA Access Library Prep protocol:
For more information, see the reference guide.
Starting from total RNA input, it takes two days for 8–16 samples and three days for 17–48 samples until libraries are ready to load on the flow cell. This includes approximately 11 hours of hands-on time.
This kit is similar to TruSeq Stranded Total RNA Library Prep because it enables RNA sequencing from low quality and FFPE-derived samples, and maintains strand information. Unlike TruSeq Stranded Total RNA Library Prep, this kit only captures the coding regions of the transcriptome, allowing much higher throughput and requiring lower sequencing depth.
This kit is similar to TruSeq Stranded mRNA Library Prep because it offers sequencing of the coding RNA and does not require ribosomal depletion.
This kit is compatible with low quality and FFPE-derived samples because it does not rely on the presence of a poly-A tail.
This kit is available in a Set A and a Set B, each containing 12 indexes. When used together, sets A and B provide a total of 24 unique indexes.
Use Agilent Technologies Human UHR total RNA (catalog # 740000) as a control sample for this protocol. UHR contains the following known fusions: BCR-ABL1, BCAS3-BCAS4, and NUP214-XKR3 (at low levels).
Quantify the library using a Fragment Analyzer or Bioanalyzer. Alternatively, use PicoGreen.
Since it enriches the coding transcriptome, the ribosomal RNA is washed away during the wash procedures.
When used together, TruSeq RNA Single Index Sets A and B allow for pooling up to 24 samples using the 12 different indexes in each kit.
Use 200 ng of each RNA library.
You can prepare one sample at a time.
The size of the final product is ~250–300 bp. A larger fragment size is expected for good FFPE RNA (> 350 nt), while a smaller fragment size is expected for poor FFPE RNA.
Assess the final library quality with either an Advanced Analytical Technologies Fragment Analyzer using a NGS Fragment Analysis Kit or Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip.
Use qPCR to quantify libraries. For more information, see the Sequencing Library qPCR Quantification Guide.
All Illumina sequencing platforms except HiSeqX.
The standard sequencing primers included in the cluster generation kits are required.
The recommended read length is 2 x 75 bp.
BaseSpace Sequence Hub core apps for RNA include the industry standard TopHat and Cufflinks analysis pipeline. Additional, third-party analysis tools are also available.
Various data files and plots comprise the output from analyzing TruSeq RNA Access libraries. For details, see BaseSpace Core Apps for RNA Sequencing.