rna seq for illumina sequencing

A unique workflow adds partial adapters directly to RNA to retain small RNA, coding RNA, and noncoding RNA to sequence the entire transcriptome.

  • <6.5 hours for rRNA depletion and library generation
  • Ribosomal depletion module is compatible with human, mouse, and rat samples
  • For highest success rates, tracking dyes in each reagent change color when properly mixed

Retain transcriptome diversity

The Collibri Stranded RNA Library Prep Kit for Illumina Systems with Human/Mouse/Rat rRNA Depletion uses a unique protocol in which adapters are added directly to RNA. The full transcriptome is retained because cDNA priming is not affected by GC content and random oligonucleotide sequences are not incorporated into cDNA, preventing false SNPs and point mutations.

  Collibri Stranded RNA Library Prep Kit for Illumina
with H/M/R rRNA Depletion Kit
Hands-on time (hr) 3.5
Total time (hr) 6.5
RNA source Total RNA
Input range (ng) 100–1000
Species compatibility Human, mouse, rat
Ribosomal depletion module Included
Analysis difficulty Biologist-friendly

Visual feedback at each step in the workflow

To achieve the highest success rate, tracking dyes change color at each step only when proper mixing is achieved. Lack of a color change indicates the need to pause and complete mixing prior to continuing. Visual feedback is useful to monitor the performance of robotic systems.

Video: Collibri NGS Library Prep Kits for Illumina systems

Collibri NGS library prep kits contain unique tracking dyes in each reagent to show in real time if samples are properly mixed.

Tips for your RNA-Seq library preparation

The scientists who developed the Collibri Stranded RNA Library Prep kits share their tips for cDNA library generation that retains the full transcriptome.

Tips on rRNA depletion
Tips on enzymatic RNA fragmentation
Tips on adapter hybridization, adapter ligation, and reverse transcription
Ten quick tips on cleanup
Tips on using the PCR index primers plate
Colored dye benefits

Recommended controls

Invitrogen ERCC RNA Spike-In Control Mix (not included in the Collibri kit) is recommended to be added to the input RNA before library preparation. It  provides a set of external RNA controls that enable performance assessment (dynamic range, lower limit of detection, fold-change response) of a variety of technology platforms used for gene expression experiments. For detailed information, see the ERCC RNA Spike-In Control Mixes User Guide.

Transcript molar ratios in ERCC Spike-In Mixes

Figure 2. Transcript molar ratios in ERCC Spike-In Mixes. The transcripts in Spike-In Mix 1 and Spike-In Mix 2 are present at defined Mix 1:Mix 2 molar concentration ratios, described by four subgroups. Each subgroup contains 23 transcripts spanning a 106-fold concentration range, with approximately the same transcript-size distribution and GC content.

For a positive RNA control, we recommend Universal Human Reference RNA or Human Brain Total RNA.

Tip: Do not use the ERCC RNA Spike-In Control Mix with highly degraded samples such as FFPE RNA. High quality ERCC RNA can out-compete lower quality RNA populations, causing ERCC RNA to be over-represented in the final library. 


Whole transcriptome library preparation workflow

Retain coding and noncoding RNA diversity

We offer a range of Invitrogen genomic RNA extraction kits for sensitive, scalable purification from an expansive set of starting materials to help maximize process efficiency and downstream performance. This includes a broad range of kits for purifying RNA from a variety of samples including tissue, cells, blood, serum, plants, forensic samples, and more.
Automate RNA extraction using magnetic bead-based technology and KingFisher instruments to bind, wash, and elute RNA to reduce your hands-on time while maintaining high yields and excellent reproducibility.

Quantification options to scale throughput

The qPCR-based Collibri Library Quantification kit scales well for larger sample batches and is the ideal method for precious samples or clinical samples. Quantification accuracy is equivalent to the KAPA Library Quantification kit with the additional benefit of visual feedback during the quantification process.

The Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample. 

Regardless of the assay that is chosen, good laboratory technique should be used in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.

Recommended accessories

With Applied Biosystems QuantStudio real-time PCR systems, you get true value with excellent performance, reliability, and world-class support. Our family of instruments enables you to obtain the results you need, connect and collaborate with colleagues, and achieve your research goals.

Retain coding and noncoding RNA diversity

We offer a range of Invitrogen genomic RNA extraction kits for sensitive, scalable purification from an expansive set of starting materials to help maximize process efficiency and downstream performance. This includes a broad range of kits for purifying RNA from a variety of samples including tissue, cells, blood, serum, plants, forensic samples, and more.
Automate RNA extraction using magnetic bead-based technology and KingFisher instruments to bind, wash, and elute RNA to reduce your hands-on time while maintaining high yields and excellent reproducibility.

Quantification options to scale throughput

The qPCR-based Collibri Library Quantification kit scales well for larger sample batches and is the ideal method for precious samples or clinical samples. Quantification accuracy is equivalent to the KAPA Library Quantification kit with the additional benefit of visual feedback during the quantification process.

The Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample. 

Regardless of the assay that is chosen, good laboratory technique should be used in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.

Recommended accessories

With Applied Biosystems QuantStudio real-time PCR systems, you get true value with excellent performance, reliability, and world-class support. Our family of instruments enables you to obtain the results you need, connect and collaborate with colleagues, and achieve your research goals.