Description

CORALL Total RNA-Seq Library Prep Kit

The CORALL Total RNA-Seq Library Prep Kit enables fast and cost-efficient generation of UMI labelled, stranded libraries for whole transcriptome analyses using Illumina® NGS platforms.

Transcript Coverage

CORALL generates transcriptome-wide smooth, uniform read coverage, comparable to that of competitor kits (Fig. 1).

CORALL_Figure_01

Figure 1 | Accumulated transcript body coverage (whole transcriptome). Coverage across all transcripts was generated using the geneBody_coverage.py tool provided by RSeQC (transcripts length normalized to 100 %).

Gene Detection

CORALL delivers excellent gene discovery rates matching conventional competitor kits (Fig. 2A). 95% of genes are commonly detected between CORALL and each of the two competitors (at >10 counts per million (CPM), Fig. 2B). This high level of library complexity ensures faithful representation of the transcriptome, enabling sensitive expression profiling.

CORALL_Figure_02_A_B

Figure 2 | Gene detection. A) Gene discovery rates. The number of detected genes is plotted against the total number of reads mapping uniquely to exons (calculated with featureCounts). B) Overlap of detected genes. The Venn diagram illustrates overlaps between CORALL and competitor kits for genes detected with normalized expression levels >10 CPM (for uniquely mapping reads).

Superior End-to-End Coverage

CORALL’s comprehensive coverage delivers improved transcript start and end site representation. Read coverage was analyzed using the ERCC spike-in controls, which feature precise, known transcription start and end sites (TSS and TES, respectively). CORALL reads map more accurately to the exact ERCC TSS (Fig. 3A) than competitor libraries, which fail to cover the true start sites. Additionally, CORALL provides elevated coverage at TES (Fig. 3B).

Figure 3 | Normalized ERCC coverage of A) TSS and B) TES. Normalized coverage of accumulated mapped reads for all detected ERCCs. The absolute nucleotide positions relative to the TSS (red dotted line, A) and TES (blue dotted line, B) are shown.

Flexible Input

CORALL libraries can be prepared from poly(A)-enriched or rRNA-depleted RNA (100 – 200 ng of total RNA yields approximately 1 – 2 ng of rRNA-depleted RNA). We recommend using Lexogen’s RiboCop rRNA Depletion Kit V1.2 (Cat. No. 037) or Poly(A) RNA Selection Kit (Cat. No. 039). Total RNA without prior depletion or enrichment (100 pg to 100 ng) can also be used as input.

Unique Molecular Identifiers (UMIs) seamlessly included

UMIs are seamlessly introduced into CORALL libraries as an inherent part of the protocol. As a result Read 1 contains the UMI information making it directly accessible in the cost-efficient single-read mode.

Enhanced Multiplexing

CORALL libraries can be multiplexed using up to 96 i7 indices (included in the kit). Additional i5 indices can also be introduced using the Lexogen i5 6 nt Dual Indexing Add-on Kits (Cat. No. 047). Used together, Lexogen’s 96 i7 and 96 i5 indices enable up to 9,216 different index combinations for sequencing.

Fast and Easy All-in-One Protocol

The streamlined RNA fragmentation-free protocol generates ready-to-sequence libraries in 4.5 hours.

Workflow

Reverse Transcription
Step 1:
CORALL Total RNA-Seq Library Prep uses total, poly(A) enriched
or rRNA depleted RNA as input.
corall_workflow_01_V2timer_corall_v2
Reverse Transcription
Step 1:
CORALL library generation is initiated by random hybridization
of Displacement Stop Primers (DSP) with partial Illumina-compatible
P7 sequences, to the RNA template. No prior RNA fragmentation is
necessary, as the insert size is determined by the distance between
hybridized DSPs. Reverse transcription extends each DSP to the next,
where transcription is effectively stopped. This stop prevents spurious
second strand synthesis, maintaining excellent strand specificity.
corall_workflow_02_V2timer_corall_v2
Linker Ligation
Step 2:
Highly efficient ligation of Linker Oligos to the 3’ ends of first-strand
cDNA fragments then introduces partial Illumina-compatible P5
sequences and Unique Molecular Identifiers (UMIs).
timer_corall_v2
Library Amplification
Step 3:
During PCR, second strand synthesis is performed, and the
double-stranded cDNA is amplified. In doing so, i7 and (optional)
i5 indices as well as complete adapter sequences required for
cluster generation on Illumina instruments are added.
timer_corall_v2
Sequencing
Step 4:
The UMI is positioned to be read out at the beginning of Read 1
during sequencing and can therefore be assessed even in Single-Read
mode. All purification steps are based on magnetic beads, rendering
the protocol highly suitable for automation.
corall_workflow_05_V2timer_corall_v2

FAQ

Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.

CORALL has broad species compatibility. Libraries have been successfully generated using: human (including whole blood RNA and leukocyte RNA), mouse, bacterial, plant, hamster, and mini pig RNA samples.
Any input amount between 1ng and 1 µg of total RNA for rRNA depletion or poly(A) enrichment, or up to 100 ng input for total RNA without enrichment/depletion can be used.
We do not recommend multiplexing Lexogen libraries with libraries from other vendors in the same sequencing lane.

Though this is possible in principle, specific optimization of index combinations, library pool¬ing conditions, and loading amounts may be required, even for advanced users. Sequencing complex pools that include different library types at different lane shares may have unpredict¬able effects on sequencing run metrics, read quality, read outputs, and / or demultiplexing per¬formance. Lexogen assumes no responsibility for the altered performance of Lexogen libraries sequenced in combination with external library types in the same lane (or run).

Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq, QuantSeq-Flex, SENSE, or CORALL libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc), which are provided in our Library Prep Kit User Guides, and online Frequently Asked Questions (FAQs).

CORALL libraries can be sequenced either with single-read (SR) or paired-end (PE) sequencing. We can recommend SR50 – 200, or PE100 – 150. The choice of sequencing format may also depend on the particular application.
CORALL libraries can be analyzed using most standard RNA-Seq pipelines. The ideal workflow may depend on the goal of the sequencing experiment. Further information on recommendations for trimming, quality control, and alignment of CORALL reads can be found in the Data Analysis section of the CORALL product page, or in the CORALL User Guide, Appendix H, p.34.
Please follow loading amount recommendations provided by Ilumina for your specific instrument, or contact info@lexogen.com for further information.

Downloads

CORALL Total RNA-Seq Library Prep Kit for Illumina

pdf User Guide – Release 28.02.2019
pdf Product Flyer
pdf PCR Add-on Kit for Illumina Instruction Manual – update 08.08.2018
pdf i5 Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 24.05.2018 (release of additional i5 Unique Dual Indexing Add-on Kit (5001-5096))

pdf Lexogen i7 and i5 Index Sequences – including for kits bought before 17.02.2017
pdf Library Quantification Calculation File

Material Safety Datasheets

pdf MSDS information for CORALL Total RNA-Seq Library Prep Kit for Illumina

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

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If you would like to receive a CORALL Total RNA-Seq Free Trial Kit, please fill in the form.

If you have any questions, please do not hesitate to contact us at info@lexogen.com or +43 (0) 1 345 1212 – 41.

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