RNA Library Preparation and Sequencing | High-Throughput Sequencing Facility (HTSF)

Total RNA-seq

Whole-transcriptome analysis with total RNA sequencing (RNA-Seq) detects coding plus multiple forms of noncoding RNA. Total RNA-Seq can accurately measure gene and transcript abundance and identify known and novel features of the transcriptome.

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	Tecan Genomics Ovation Universal RNAseq with any deplete Nugen Solo Nugen Trio

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	NovaSeq S4	2×50
	38	NovaSeq S2
	16	NovaSeq S1
	8	NovaSeq SP
	3	HiSeq 4000
	2	HiSeq 4000

*NOTE: Anticipated yield of 100 million clusters (200 million reads) per library

mRNA-Seq

mRNA sequencing (mRNA-Seq) has rapidly become the method of choice for analyzing the transcriptomes of disease states, of biological processes, and across a wide range of study designs. mRNA-Seq can identify both known and novel transcript isoforms, gene fusions, and other features as well as allele-specific expression. mRNA-Seq delivers a complete view of the coding transcriptome that is not restricted by the filter of prior knowledge.

There are a variety of approaches to mRNA sequencing. Therefore, the best approach will depend on your needs.
Sciclone 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	HiSeq 4000

*NOTE: Anticipated yield of 50 million clusters (100 million reads) per library

Small RNA sequencing

MicroRNA sequencing (miRNA-seq), a type of RNA-Seq, is the use of next-generation sequencing to sequence microRNAs, also called miRNAs. miRNA-seq differs from other forms of RNA-seq in that input material is typically enriched for small RNAs. miRNA-seq allows researchers to examine tissue-specific expression patterns, disease associations, and isoforms of miRNAs, and to discover previously uncharacterized miRNAs.

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

*NOTE: Anticipated yield of 20 million reads/clusters per library

Relevant RNA Services Forms and Guides

HTSF Sample Preparation Requirement Guide
Illumina Platform Comparison and Specification Table
NovaSeq Requirements
NovaSeq White Paper
Approved Containers Information