Collibri Library Prep Kits for mRNA-seq on Illumina Systems

* 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.

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.

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.

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.

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.

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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.

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.