Description

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.

Unparalleled Sensitivity

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.

Method Input Detected Genes*
LUTHOR 3’ mRNA-Seq 1 cell (HEK293T) ~11,000 – 15,000
SMART-Seq v4 3’ DE 1 cell (K562) ~ 6,000 – 8,0001
SMART-Seq Single Cell (WTS) 1 cell (GM22601) ~9,000 – 10,5001
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 [1] 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.

[1] https://www.takarabio.com/,  (2020) TECH NOTE: Unprecedented sensitivity with the SMART-Seq Single Cell Kit.
[2] 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
[3] 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).

LUTHOR New Detected Genes

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.

High Strand-Specificity

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).

LUTHOR Mapping Class Attributions

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 support@lexogen.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.

Workflow

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.

Input
Step 1:
Single, e.g. FACS-sorted animal cells or ultra-low input RNA
(10 pg -1 ng)
LUTHOR_Workflow_011-3_Timer_Workflow_LUTHOR
THOR Amplification: Oligo(dT) Priming
Step 1:
THOR (T7 High-resolution Original RNA) Amplification is initiated by
oligo(dT) priming. The primer contains a partial Illumina-compatible
P7 linker (blue) and a T7 promoter sequence (light brown).
LUTHOR_Workflow_021-3_Timer_Workflow_LUTHOR
THOR Amplification: Reverse Transcription
Step 1:
Reverse transcriptase elongates the primer and generates
cDNA to stabilize the RNA template.
1-3_Timer_Workflow_LUTHORLUTHOR_Workflow_03
THOR Amplification: End Repair
Step 2:
The proprietary end repair step removes the single-stranded
3’ poly(A) overhang and generates a double stranded
T7 promoter sequence for RNA amplification.
1-3_Timer_Workflow_LUTHORLUTHOR_Workflow_04
THOR Amplification: in vitro Transcription
Step 3:
LUTHOR_Workflow_05
During an overnight in vitro transcription step the original
mRNA template is copied repeatedly by linear amplification.
1-3_Timer_Workflow_LUTHOR
THOR Amplification: in vitro Transcription (overnight)
Step 3:
LUTHOR_Workflow_06
Antisense-RNA copies are generated which contain the partial
Illumina compatible P7 linker at the 5’ end.
1-3_Timer_Workflow_LUTHOR
Library Generation: Reverse Transcription
Step 4:
LUTHOR library generation is initiated by random priming using
Lexogen’s proprietary Displacement Stop technology. Random
primers contain partial Illumina-compatible P5 sequences (green).
LUTHOR_Workflow_074_Timer_Workflow_LUTHOR
Library Amplification: PCR
Step 5:
During PCR, second strand synthesis is performed, and the
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.
LUTHOR_Workflow_085_Timer_Workflow_LUTHOR
Sequencing
Step 6:
LUTHOR contains the Read 1 linker sequence in the random primer,
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.
LUTHOR_Workflow_09

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.

LUTHOR is differentiated by the fact that it utilizes a pre-amplification step, where the libraries are generated from amplified RNAs. This direct RNA copy enables the generation of libraries from ultra-low input RNA, down to the single-cell level. RNA amplification in LUTHOR is templated by the endogenous mRNA molecules instead of using cDNA intermediates.

QuantSeq utilizes a different chemistry where no pre-amplification step is employed, requiring higher RNA input amounts. QuantSeq captures the 3’ end of transctipts by oligo(dT) priming and reverse transcription. During reverse transcription a partial Illumina compatible adapter is introduced at the 3’ end. After removal of the template RNA, the second strand is generated by random priming and extension using a DNA polymerase.

LUTHOR can be used for direct RNA amplification from animal cells or protoplasts (e.g., from plants), or with purified RNA from all organisms and viruses that possess poly(A)-tailed RNA species. LUTHOR is not suitable for analysis of non-polyadenylated RNA, e.g., total RNA from bacterial species.

Input ranges:

  • FACS-Sorted Cells: 1 – 100 cells per well/sample may be used. For sorting of single cells, we recommend to sort directly into 5 µl Cell Lysis Buffer (CLB).
  • Cell suspensions: 1 – 100 cells/sample in a volume of 2 µl. Prepare cell suspension in 1x PBS.
  • Purified RNA: 10 pg – 1 ng in a volume of 4 µl.

Higher inputs are possible and require minor protocol modifications. For more information please contact Lexogen Tech Support at support@lexogen.com.

No, we recommend the QuantSeq FWD 3’ mRNA-Seq Library Prep Kit for degraded RNA and FFPE sample types. FFPE material is very heterogenous and highly variable in terms of quality, degree of cross-linking, and mRNA accessibility, which may lead to insufficient library generation.

Most steps in the LUTHOR protocol rely on master mixes of reaction components including enzymes. Therefore, the reaction steps can be automated on standard liquid handlers. LUTHOR uses 4 purification steps during the procedure. The purification procedures use a limited amount of magnetic beads, therefore, purification steps may need to be optimized for automation on liquid handlers. Those interested in pursuing automation should contact support@lexogen.com.

When using cells as input for LUTHOR, special care should be taken that the cells are viable and stay intact. For best practice, cells should be kept at 4 °C prior to lysis. Dead or dying cells will result in a decrease of the data quality and may lead to an increase of reads obtained for mitochondrial transcripts. Cell suspensions or FACS-sorted cells are lysed during Step 4 of the LUTHOR protocol. It is critical that the 4 °C hold is executed. Exposure to temperatures > 4 °C at this point may lead to RNA degradation and cause a reduction in overall quality of the sequencing data, e.g., RNA degradation can lead to an increase in intronic  and rRNA reads.

Once the initial reverse transcription has occurred, LUTHOR is a highly robust protocol with little risk of generating artifacts, such as adapter dimers.

Single-read (SR) sequencing is the recommended method for LUTHOR libraries. LUTHOR contains the Read 1 linker sequence in the random primer, and so NGS reads are generated towards the poly(A) tail and directly correspond to the 3’ mRNA sequence. This results in a poly(T) stretch corresponding to the oligo(dT) primer which would begin Read 2, thus paired-end sequencing is not recommended.

The loading amounts indicated below are provided as a guideline for LUTHOR 3’ mRNA-Seq libraries in pure pools for each instrument. Multiplexing LUTHOR libraries with other external library types is not recommended (see FAQ 13). We therefore cannot provide optimal pooling or loading amount guidelines for lane mixes that include LUTHOR libraries in diverse multiplexing setups.

Loading amounts (and PhiX spike-in percentages) may still need to be adjusted to obtain optimal cluster densities for your respective instrument, or if sequencing chemistries are changed. Inaccuracies in library quantification can affect the loading amount required. Please ensure accurate quantification of lane mixes and lane mix dilutions when preparing for loading.

Loading amounts are inferred from 3’ mRNA-Seq libraries with similar features. NextSeq loading was evaluated by Lexogen. This table is subject to change and may be updated based on customer experience. For further inquiries regarding loading amounts please contact support@lexogen.com.

Sequencer LUTHOR 3’ mRNA-Seq (Cat. No. 143), PhiX: 1 – 5 %
HiSeq 3000 / HiSeq 4000 280 – 350 pM
HiSeq 2000 / HiSeq 2500 10 pM
MiSeq 6 – 15 pM
MiniSeq 1.3 – 1.8 pM
NextSeq 500 / 550 2 – 2.5 pM 1
NovaSeq 6000 Standard: 300 – 500 pM  , Xp: 325 – 400 pM

1 The values for NextSeq 500 / 550 are recommended loading amounts and have been evaluated by Lexogen to ensure optimal cluster densities of 200 – 260 K/mm2 are achieved.

No, LUTHOR 3’ mRNA-Seq is not compatible with the UMI Module for QuantSeq FWD.

We recommend a series of PCR cycles that range from typically 6 to 18, e.g., 7, 11, 14, and 17 cycles. If you are using single cells or purified RNA at the lower end of LUTHOR’s input range, you can optionally adjust the cycles to: 8, 12, 15, and 18 cycles.

We recommend using Universal Human Reference RNA (UHRR) or similar at a concentration in the range of 10 pg – 100 pg.

LUTHOR uses a unique technology to generate libraries. The library size distribution is broad and comparable to CORALL.
LUTHOR BA trace input gradient A

Figure 1 | Bioanalyzer traces of LUTHOR libraries prepared from different input amounts of purified total Universal Human Reference RNA (UHRR). Libraries were prepared using 10 pg (red trace, 11 PCR cycles), 25 pg (blue trace, 9 PCR cycles), 50 pg (green trace, 9 PCR cycles), and 100 pg (cyan trace, 8 PCR cycles) of UHRR input. Endpoint PCR was performed using i5 / i7 dual indexing.

LUTHOR sequencing data can be analyzed using a standard 3’ mRNA-Seq data analysis pipeline, including steps for Trimming, Mapping, and Counting. Please contact support@lexogen.com for further information on how to analyze your LUTHOR 3’ mRNA-Seq data.

We do not recommend multiplexing Lexogen libraries with other library types in the same sequencing lane. Though this is possible in principle, specific optimization of index combinations, library pooling 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 unpredictable effects on sequencing run metrics, read quality, read outputs, and/or demultiplexing performance. 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 LUTHOR, QuantSeq, 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).

Lexogen UDI 12 nt Unique Dual Indexing Sets (Set A: Cat. Nos. 101 – 104 and 156; Set B: Cat. No. 105) are recommended for use with LUTHOR libraries. The maximum multiplexing capacity for LUTHOR libraries with the Lexogen UDI 12 nt Indices is 384.

Indices from other vendors are compatible with LUTHOR only if they utilize the Illumina TruSeq partial adapters.

We recommend targeting a read depth of 1 – 5 M reads/sample. Maximal multiplexing capacity is thus approximately 10 x higher than for conventional whole transcriptome mRNA-Seq.

Yes, and we recommend a PBMC selection to minimize the effect of globin on the data output.

Downloads

LUTHOR 3’ mRNA-Seq Library Prep Kit for Illumina

pdf User Guide – release 22.10.2020
pdf Technical Note – update 12.11.2020

Material Safety Datasheets

MSDS Information can be found in the Documents page.

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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