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LUTHOR 3’ mRNA-Seq Library Prep Kit for Illumina
LUTHOR combines a novel direct RNA amplification technology with an efficient one-step 3’ RNA-Seq library preparation method yielding unprecedented sensitivity and reproducibility for individual cells and purified RNA in the ultra-low pg range.
LUTHOR 3’ mRNA-Seq offers unprecedented sensitivity for ultra-low input RNA and single cells compared to market leading commercial and academic protocols (Tab. 1).
Table 1 | LUTHOR provides unprecedented sensitivity.
|LUTHOR 3’ mRNA-Seq||1 cell (HEK293T)||~11,000 – 15,000|
|SMART-Seq v4 3’ DE||1 cell (K562)||~ 6,000 – 8,0001a|
|SMART-Seq Single Cell (WTS)||1 cell (GM22601)||~9,000 – 10,5001b|
|CEL-Seq 2 (C1 HT) (WTS)**||1 cell (mES)||~7,0002|
|SMART-Seq 2 (WTS)**||1 cell (mES)||~8,000 – 9,0002|
|SMART-Seq 3 (WTS)**||1 cell (HEK293FT)||~11,000 – 12,0003|
*Obtained from manufacturers specifications  and from published benchmarking studies [2,3] at 1 M reads / sample, threshold of >1 Counts Per Million (CPM). Methods are shown for 3’-Seq and whole transcriptome sequencing (WTS). **Academic protocol.
 https://www.takarabio.com/, a TECH NOTE: Differential expression from single cells using the SMART-Seq v4 3′ DE Kit (2020), b TECH NOTE: Unprecedented sensitivity with the SMART-Seq Single Cell Kit (2020).
 Ziegenhain, C., Vieth, B., Parekh, S., Reinius, B., Guillaumet-Adkins, A., et al., 2017. Comparative Analysis of Single-Cell RNA Sequencing Methods. Mol Cell, 65:631-643.e4, DOI: https://doi.org/10.1016/j.molcel.2017.01.023
 Hagemann-Jensen, Ziegenhain, C., Chen, P., Ramsköld, D., Hendriks, G.-J., et al., 2020. Single-cell RNA counting at allele- and isoform-resolution using Smart-seq3. Nat Biotechnol. 38: 708–714, DOI: https://doi.org/10.1038/s41587-020-0497-0
LUTHOR reliably detects ~13,000 to 16,000 genes from ultra-low input RNA (10 – 100 pg) and ~6000 – 12,500 genes per frozen single cell at 1 M reads / sample (Fig.1).
Figure 1 | LUTHOR 3’ RNA-Seq enables highly sensitive and consistent gene detection already at low sequencing depth. A) LUTHOR libraries were generated from ultra-low input Universal Human Reference RNA (UHRR, 4 replicates) or B) from frozen single human HEK293T cells, single mouse embryonic stem (mES) cells, and single Drosophila S2 cells (8 replicates per cell type). Libraries were sequenced, and detected genes were analyzed for 1 M reads / sample. The number of detected genes was counted at a threshold of >1 Counts Per Million (CPM). The box denotes the interquartile range (IQR), i.e., the 25th and 75th quartiles; the whiskers represent the extremes of the data.
LUTHOR maintains exceptional strand-specificity of >99.9 % and allows to map reads to their corresponding strand on the genome, enabling the discovery and quantification of antisense transcripts and overlapping genes.
Direct Counting for Gene Expression Quantification
Just one fragment per amplified antisense RNA copy is produced; therefore, no length normalization is required. This allows straightforward determination of gene expression values even at single cell level.
High Quality Performance for Ultra-low Input and Single Cells
LUTHOR 3’ mRNA-Seq reliably represents endogenous mRNA composition and efficiently excludes ribosomal rRNAs focusing sequencing reads on coding sequences (Fig. 2).
Figure 2 | Feature distribution of uniquely mapped reads for LUTHOR 3’ mRNA-Seq. The majority of LUTHOR reads generated from 10 and 100 pg UHRR, or 1 and 10 single HEK293T cells map to exonic sequences.
Excellent Replicate and Cell-to-Cell Reproducibility
The combination of innovative THOR Amplification Technology and robust library generation delivers excellent reproducibility for replicates of ultra-low input RNA (Fig. 3A) and even for individual mES cells after freezing (Fig. 3B).
Figure 3 | Excellent reproducibility for LUTHOR 3’ mRNA-Seq. A) Correlation plot of gene counts for replicates from 10 pg UHRR and B) from two individual FACS-sorted mES cells at 1 M reads / sample. Cells were frozen after sorting and stored at -80 °C prior to processing with LUTHOR 3’ mRNA-Seq.
Cost Saving Multiplexing
LUTHOR 3’ mRNA-Seq library preps are intended to be used with Unique Dual Indices (UDIs). Lexogen offers 12 nt Unique Dual Indexing Sets with up to 384 pre-mixed UDIs in a convenient 96-well plate format. Lexogen’s new 12 nt UDIs feature superior error correction for maximal sequencing data output and are now also available as stand-alone Unique Dual Indexing Sets (Cat. No. 101 – 105, 156) for use with LUTHOR library preps. For further questions, please contact email@example.com.
This high level of multiplexing allows saving sequencing costs, especially in combination with shorter read lengths. LUTHOR is also designed to yield insert sizes suitable for Single Read (SR) 50 to SR150.
Mapping of Transcript End Sites
LUTHOR libraries contain inserts with an average size of ~150 base pairs. By using longer reads LUTHOR therefore allows to pinpoint the exact 3’ end of poly(A) RNA within single cells and to obtain accurate information about the 3’ UTR.
Simple Bioinformatics Analysis
Read mapping is simplified by skipping the junction detection. Reads are generated at the transcripts’ 3′ end where nearly no junctions are located. Data processing can hence be accelerated.
“We have used the LUTHOR 3’ mRNA-Seq Library Prep Kit to profile single cells, which we FACS sorted before. The quality of the data is impressive: we detect high numbers of transcripts per cell by moderate sequencing depth. This allows us to comprehensively identify cell state and subtle cell state changes in the analyzed samples. For these aims, the 3’ mRNA-Seq library generated with the LUTHOR Kit has advantages over full-length cDNA approaches we have used previously. I can recommend LUTHOR for single-cell RNA-Seq analysis.”
LUTHOR 3’ mRNA-Seq uses Lexogen’s proprietary THOR (T7 High-resolution Original RNA) Amplification Technology to enable true high-resolution sequencing of single cells with unprecedented sensitivity. During the THOR procedure, RNA is amplified directly from the original mRNA pool of a single cell. Using a proprietary step, a T7 promoter is fused to the endogenous mRNA molecules and RNA-templated in vitro transcription generates antisense RNA copies. The RNA copies are subsequently converted to 3’ mRNA-Seq libraries in a single step (see workflow). The complete LUTHOR workflow is free from ligation, template switch, and fragmentation and thus also supports challenging samples.
(10 pg -1 ng)
oligo(dT) priming. The primer contains a partial Illumina-compatible
P7 linker (blue) and a T7 promoter sequence (light brown).
cDNA to stabilize the RNA template.
3’ poly(A) overhang and generates a double stranded
T7 promoter sequence for RNA amplification.
mRNA template is copied repeatedly by linear amplification.
Illumina compatible P7 linker at the 5’ end.
Lexogen’s proprietary Displacement Stop technology. Random
primers contain partial Illumina-compatible P5 sequences (green).
double-stranded cDNA is amplified. In doing so, i7 and i5 indices
as well as complete adapter sequences required for cluster
generation on Illumina instruments are added.
hence NGS reads are generated towards the poly(A) tail and directly
correspond to the mRNA sequence. LUTHOR libraries should be
sequenced in Single Read (SR)-mode. A poly(T) stretch corresponding
to the oligo(dT) primer is located at the beginning of Read 2, therefore
paired-end sequencing is not recommended.
Frequently Asked Questions
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LUTHOR 3’ mRNA-Seq Library Prep Kit for Illumina
Technical Note – update 12.11.2020
Material Safety Datasheets
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