Library Preparation
DNA Sequencing RNA Sequencing Methylation Sequencing Library Preparation High-Throughput Sequencing

RNA library preparation

Simple, customized RNA-Seq workflows with highly accurate results

Advances in RNA-Seq library prep are revolutionizing the study of the transcriptome

Whole transcriptome

Measure gene and transcript abundance; detect known and novel features in coding and noncoding RNA. Targeted hybridization removes abundant rRNA to focus on high-value portions of the transcriptome.

mRNA

Quantify gene expression, identify known and novel isoforms in the coding transcriptome, detect gene fusions, and measure allele-specific expression.

RNA enrichment

Analyze gene expression in a focused set of genes of interest. Provides quantitative expression information as well as the detection of small variants and gene fusions.

RNA amplicon

Use ultra-deep sequencing of polymerase chain reaction (PCR) amplicons for analysis of RNA sequences of interest. Perform differential expression analysis, allele-specific expression measurement, and gene fusion verification.

Pipetting close up

RNA sequencing methods guide

This guide provides solutions for profiling RNA, from targeted panels to the whole transcriptome. Illumina RNA-Seq workflows integrate library prep, sequencing, and data analysis to support transcriptome research.

Choosing an RNA library prep kit for your experiment

Product Benefits
Single-cell RNA sequencing
Illumina Single-Cell 3’ RNA Prep
  • Accessible and highly scalable single-cell RNA-Seq solution for mRNA capture, barcoding, and library prep
  • Does not require expensive microfluidic equipment or labor-intensive protocols
  • Wide processing range from hundreds to hundreds of thousands of cells to reveal rare cell types
  • Run one sample or up to 96 samples in parallel with sample multiplexing
  • Compatible with custom single-cell measurements for expanded multiomic applications
mRNA Sequencing 
Illumina Rapid mRNA Prep
  • Fastest mRNA‑Seq prep in the Illumina portfolio using a single tube reaction
  • Simplified workflow combines cDNA synthesis and on‑bead tagmentation to reduce hands‑on time
  • Low-input flexibility supports 1–1000 ng RNA
  • Efficient polyA capture for focused, high‑confidence gene expression analysis
  • Accurate, reproducible results with nonstranded libraries and uniform coverage
Illumina Stranded mRNA Prep
  • Cost-effective, scalable RNA sequencing of coding transcriptome with precise measurement of strand orientation
  • Broad RNA input range, cost-effective sequencing with up to 384 UDIs
  • Wide dynamic range for more accurate measurement of gene expression
  • Poly(A) capture, ligation-based addition of adapters and indexes
Total RNA Sequencing
Total RNA Sequencing
  • Fastest total RNA‑Seq prep in the Illumina portfolio with a streamlined, single tube reaction
  • Simplified workflow combines cDNA synthesis and on‑bead tagmentation to reduce hands‑on time
  • Low input flexibility supports 1–1000 ng RNA, > 20ng for FFPE samples
  • Broad transcriptome coverage enabled by efficient rRNA and globin RNA depletion
  • High‑accuracy, reproducible data for nonstranded whole‑transcriptome analysis
Illumina Stranded Total RNA Prep
  • Integrated enzymatic RNA depletion removes both rRNA and globin mRNA in a single, rapid step
  • One tube to deplete abundant transcripts from multiple species provides flexibility for mixed samples
  • Use with human, mouse, rat, bacteria and epidemiology samples
  • Broad RNA input range, cost-effective sequencing with up to 384 unique dual indexes (UDIs)
  • Ligation method results in high coverage uniformity and reliability even from degraded samples
  • Measure fold changes in FFPE and low-quality samples
Targeted RNA Sequencing
Illumina Rapid RNA with Exome 2.5 Enrichment
  • Fast RNA exome workflow with a single-tube cDNA synthesis and tagmentation reaction
  • Focused coverage of the coding transcriptome using the Exome 2.5 enrichment panel
  • Low-input flexibility supports 1-1000 ng RNA and 20-1000 ng FFPE RNA
  • High-quality data for gene expression, fusion detection, and isoform analysis
Illumina Rapid RNA Enrichment
  • Fastest tagementation-based workflow for targeted RNA-Seq with < 1.5 hours hands-on time tagmentation
  • Low-input flexibility supports 1–1000 ng RNA, >20ng for FFPE samples
  • Enables high-quality data for gene expression, fusion detection, small variant detection, and splicing isoform analysis
  • Customizable enrichment panels for human RNA applications
  • Concierge services for non-human targeted RNA enrichment panel designs`
Illumina RNA Prep with Enrichment
  • Tagmentation-based library prep enables deep insights into many transcripts of interest, including the RNA exome
  • Single hybridization step
  • No mechanical shearing needed
  • Compatible with low quality / degraded / FFPE tissue
Illumina miRNA Prep
  • Gel-free workflow reduces sample loss and simplifies library preparation
  • Optimized adapter chemistry minimizes adapter dimers and improves miRNA detection
  • Unique Molecular Identifiers (UMIs) enable more accurate miRNA quantification
  • Supports low-input samples with as little as 1 ng RNA and multiplexing up to 384 UDIs

Overall the high performance, flexibility and streamlined single-day workflow make these kits an ideal solution for RNA prep.

Dr. Christopher Noune
 Australian Genome Research Facility

RNA library prep at-a-glance

  Illumina Rapid Total RNA Prep Illumina Rapid mRNA Prep Illumina Rapid RNA Prep with Enrichment
Application
Whole transcriptome
 mRNA RNA enrichment
Hands-on time < 1.5 hrs < 1.5 hrs < 1.5 hrs
Turnaround time ~ 3.5 hrs 3 hrs 6.5 hrs
Input 1 to 1000 ng standard quality RNA; >20 ng for FFPE 1 to 1000 ng standard quality RNA 1 to 1000ng standard quality RNA, > 20ng FFPE
Automation capability Liquid handling robots Liquid handling robots Liquid handling robots
PCR protocol Yes Yes Yes
Library Quant needed? Yes Yes Yes
Fragmentation included? Yes Yes Yes

All three kits allow you to decrease sequencing costs by loading up to 384 samples per flow cell or per lane on NovaSeq instruments using 384 unique dual indexes for higher throughput sequencing.

  Illumina Stranded Total RNA Prep Illumina Stranded mRNA Prep Illumina RNA Prep with Enrichment Illumina Single-Cell 3’ RNA Prep
Application
Whole transcriptome
 mRNA RNA enrichment Single-cell RNA sequencing
Hands-on time < 3 hrs < 3 hrs < 2 hrs 10 mins
Turnaround time ~7 hrs 6.5 hrs < 9 hrs ~ 15 hrs
Input 1 to 1000 ng standard quality RNA; 10 ng for optimal performance and FFPE samples 25 to 1000 ng standard quality RNA 10 ng standard quality RNA; 20 ng RNA for low quality / degraded / FFPE 100 to 200,000 cells
Automation capability Liquid handling robots Liquid handling robots Liquid handling robots N/A
PCR protocol Yes Yes Yes Yes
Library Quant needed? Yes Yes Yes Yes
Fragmentation included? Yes Yes Not required Yes
Male scientist holding a library tube in one hand and a single pipette with his other hand, actively pipetting into the tube; flow cell and SBS cartridge on the lab bench in preparation for loading; blurry image of a female scientist interacting with the touch screen monitor on a NovaSeq 6000 and library prep boxes in the background; green status bar

The benefits of tagmentation

Bead-linked transposome tagmentation is an innovative technology used in our library preparation portfolio. On-bead tagmentation lets you get to sequence-ready libraries faster than before by simultaneously fragmenting the gDNA and adding the Illumina sequencing primers. Normalize your library without ancillary reagents or equipment. Reduce turnaround time and complexity.

Learn more about tagmentation

Understanding adapter ligation

The other key technology used in our NGS library prep is adapter ligation, long known for consistent, high-quality data. Libraries are prepared by fragmenting a gDNA or cDNA sample and ligating specialized adapters to both fragment ends. These adapters contain the full complement of sequencing primer hybridization sites. This eliminates the need for additional PCR steps, making the process fully automatable.

Learn more about adapter ligation

RNA-Seq library prep methods and use cases

Tale of two kits

A critical comparison between two popular RNA library prep kits reveals new information of interest to researchers conducting RNA sequencing studies.

Building the ultimate RNA body map

Four complementary RNA-Seq methods using hundreds of cell types and tissues produced the most comprehensive atlas of the human transcriptome to date.

RNA sequencing example workflow for immuno-oncology research

1
Library prep

Illumina Rapid RNA Prep with Enrichment

A rapid, integrated workflow for producing enriched and indexed sequencing libraries from a broad range of sample types and RNA input quantities.

2
Sequencing

NextSeq 2000 System

Scalable transcriptome and exome sequencing. Process up to 40 samples in a single sequencing run.

3
Data analysis

DRAGEN RNA Pipeline

Perform secondary analysis in less than 2 hours.

Illumina Connected Multiomics

A powerful and scalable analysis environment for interpreting and visualizing multiomic data.

Related solutions

Indexing

Increase the number of samples sequenced per run and optimize high-throughput sequencing using unique dual index adapters.

UMIs

Unique molecular identifiers (UMIs) provide error correction and accuracy and can reduce false-positive variant calls while increasing variant detection sensitivity.

Automation

Our partners have developed both high- and low-throughput walk-away automation methods that span our library prep portfolio.

RNA sequencing

Detect both known and novel features in a single assay, including transcript isoforms, gene fusions, and single nucleotide variants, all without prior knowledge.

Speak to a specialist

Talk to an expert about your RNA library preparation needs.