RNA Library Preparation and Sequencing
Total RNA-seq

For library preparation recommendations based on species, please see the below chart for details.
Species | Library Preparation |
Human/Mouse/Rat | TruSeq Ribo-Zero-Gold and Kapa Ribo-Erase |
Individual species depletions |
|
For low input material, the HTSF typically recommends Tecan Genomics SOLO or Trio, which can be done with any depletion for ribosomal depletion.
General sequencing platform and pooling recommendations can be found below:
Service | Samples per Pool | Platform | Cycles |
Total RNAseq | 24 | NovaSeq S4 | 2×150 |
96 | 2×50 | ||
38 | NovaSeq S2 | ||
16 | NovaSeq S1 | ||
8 | NovaSeq SP | ||
3 | HiSeq 4000 | ||
2 |
mRNA-Seq

There are a variety of approaches to mRNA sequencing. Therefore, the best approach will depend on your needs.
Typically, the HTSF recommends the following library preparation methods:
- Kapa mRNA stranded
- Takara Smarter + Nextera XT for low input material
- Nugen mRNA can also be done by request.
Sequencing and pooling recommendations can be found in the table below:
Services | Samples per Pool | Platform | Cycles |
mRNA-Seq | 198 | NovaSeq S4 | 2×50 |
76 | NovaSeq S2 | ||
32 | NovaSeq S1 | ||
16 | NovaSeq SP | ||
6 | HiSeq 4000 | ||
4 |
Small RNA sequencing

Like other miRNA profiling technologies, miRNA-Seq has both advantages (sequence-independence, coverage) and disadvantages (high cost, infrastructure requirements, run length, and potential artifacts).
Preferred library preparation kits for this service include:
- BioO NextFlex
Sequencing and pooling recommendations can be found in the table below:
Service | Samples per Pool | Platform | Cycle |
Small RNA-seq | 48 | NextSeq 2000 | Single End/50x |
Relevant RNA Services Forms and Guides
HTSF Sample Preparation Requirement GuideIllumina Platform Comparison and Specification Table
NovaSeq Requirements
NovaSeq White Paper
Approved Containers Information