retain the entire coding transcriptome when using Collibri Stranded RNA Library Prep Kits for Illumina Systems

Generate strand-specific cDNA libraries for the study of gene expression, gene fusions, and alternative transcript isoforms by mRNA sequencing (mRNA-seq) using the Invitrogen Collibri Stranded RNA Library Prep Kit for Illumina Systems.

  • Suitable for 1–25 ng of poly(A)-enriched or rRNA-depleted RNA
  • Sequencing data reveals library complexity due to use of SuperScript IV, Dynabeads magnetic particles, and Platinum SuperFi DNA polymerase
  • Rapid 4.5-hour protocol

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 Collibri Stranded RNA Library Prep Kit for Illumina Systems
StrandedYes
Reads per sample (m)30
Total mRNA input (ng)1–25
Library prep total time (hr)4.5
Sample typeOligo(dT) enriched mRNA*
Oligo(dt) enrichment kitDynabeads mRNA DIRECT Micro Purification Kit**
Instrument compatibilityAll Illumina NGS systems
Recommended run length2 x 75 bp
Sample compatibilityAll
Recommended applications
  • Measure gene expression
  • Study gene fusions
  • Detect alternative transcript isoforms

* Previously ribo-depleted RNA may also be used to perform whole-transcriptome sequencing of non-human, mouse, or rat samples.
** Not included in Collibri Stranded RNA Library Prep Kit. Sold separately.

Rapid isolation of high purity mRNA directly from samples

The Dynabeads mRNA Direct purification kit (sold separately) contains magnetic beads for the isolation of the mRNA transcriptome from a wide variety of samples. Isolation of intact mRNA is possible thanks to RNase inhibitors in the lysis/binding buffer, combined with stringent hybridization and washing steps. Ribosomal RNA and small RNA molecules (transfer RNA, microRNA, small nucleolar RNA, and small cytoplasmic RNA) do not bind to the beads and are eliminated from the preparation. Only polyadenylated RNA species (mRNA) are captured, resulting in cleaner preparations and more sensitive results. Within minutes, pure mRNA is isolated and ready for use in downstream applications.

Sequencing data reveals library complexity

The Collibri Stranded RNA Library Prep Kit combines the superior features of SuperScript IV reverse transcriptase, Dynabeads magnetic particles, and Collibri Library Amplification Master Mix that contains Platinum SuperFi DNA polymerase to generate high-quality sequencing-ready NGS libraries that reveal the complexity of each sample.

Generate mRNA-seq libraries in 4.5 hours

Following rRNA depletion/mRNA enrichment and fragmentation, the RNA sample hybridizes to a helper Adapter Mix, which is a set of RNA/DNA oligonucleotides with a single-stranded degenerate sequence at one end and a defined sequence at the other end. The hybridized adapters are ligated to the RNA and reverse transcribed using SuperScript IV Enzyme Mix to generate cDNA. The library amplification step introduces i7 indices using Platinum SuperFi Library Amplification Mastermix and generates sequencing-ready libraries compatible with single-read or paired-end sequencing.

The Collibri Library Quantification Kit is recommended for qPCR-based quantification of libraries before sequencing.

Figure 1. The Collibri Stranded RNA Library Prep Kit ligates helper oligos, which form adapters during PCR, to single-stranded RNA to generate NGS libraries for mRNA-seq in 4.5 hours.

Visual feedback at each step in the workflow

For 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.

Improve success rates of library generation using visual feedback

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

Differentiate true gene expression and user effects

Variation in RNA-seq expression data can be attributed to a variety of factors ranging from the quality of the starting material to the person performing the experiment. The External RNA Controls Consortium (ERCC), an ad-hoc group of academic, private, and public organizations hosted by the National Institute of Standards and Technology (NIST), developed a common set of external RNA controls to distinguish between these sources of variability and true gene expression. The controls consist of a set of unlabeled, polyadenylated transcripts designed to be added to an RNA sequencing experiment after sample isolation in order to measure against defined performance criteria. Our ERCC Spike-In Mixes are pre-formulated blends of 92 transcripts, derived and traceable from NIST-certified DNA plasmids. The transcripts mimic natural eukaryotic mRNAs of 250 to 2,000 nt in length. Inclusion of ERCC controls in RNA sequencing experiments establishes a standard measure for data comparison across gene expression experiments and makes it possible to measure sensitivity (lower limit of detection) and dynamic range of an experiment.

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.

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.


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