LUTHOR HD Pool

High-Definition single-cell RNA sequencing in one tube

Down to single cells or 10 pg RNA input

Extraction-free RNA-Seq library preparation from as low as one single cell

Down to 2 EUR per library

Reduced costs with bulk configurations

Up to 36,864 samples per run

Triple-indexing allows up to 36,864 samples to be sequenced simultaneously

Up to 2,548 saved pipet tips (96 preps)

>8x less consumables with LUTHOR HD Pool than with conventional TSO-based methods

LUTHOR High-Definition Pool Single-Cell 3′ mRNA-Seq Library Prep Kit

LUTHOR HD Pool benefits from the original THOR technology to amplify minute amounts of input RNA. The Pool version includes carefully selected LPi1 sample barcodes, which are added at the very start of the library preparation. Once LPi1 barcodes are linked to each sample, pooling of 8 – 96 samples in one tube simplifies the protocol significantly reducing hands-on time and consumable costs. LUTHOR HD Pool is ideal for experiments with homogeneous sample types.

Running large scale projects?

For more samples, we recommend adding Lexogen’s patented 12 nt UDI for triple indexing (LPi1, i5, i7), and up to 36,864 different barcode combinations. Lexogen UDI Sets are available in 96-well plates and allow mixing of different Lexogen libraries (e.g., CORALL, QuantSeq, LUTHOR) with LUTHOR HD Pool libraries in sequencing lanes, contact support@lexogen.com for more information.

Scaling up? Large project? Benefit from our bulk configurations and dynamic pricing schemes! Contact us at sales@lexogen.com for a personalized quotation. When opting for bulk configurations, UDI are included in each kit.

RNA-Seq directly from lysates

LUTHOR HD Pool does not require RNA extraction. If you start from isolated cells, a dedicated lysis buffer is provided in the kit.

Sample input ranges from 10 pg (equivalent to the RNA content in one single cell) up to 10 ng. Read depth of 1 M reads / sample is usually sufficient to capture the full gene diversity, while 5 M reads / sample may be necessary for a comprehensive analysis of all transcripts (typically, there are multiple transcripts for each gene).

Add-on kits:

If you want to combine more than 96 libraries at once, we recommend our 12 nt UDI Sets.
If you prepare 7 or more pools from a single LUTHOR HD Pool Kit, you will require the PCR Add-on Kit for Illumina (Cat. No. 208)

LUTHOR HD Pool provides the same ultra-high sensitivity as the original LUTHOR HD kit with added convenience of sample pooling. It is also the ideal complement to QuantSeq-Pool for inputs < 10 ng. At Lexogen, we will always have a 3’ mRNA-Seq solution for pooled samples!

High Definition in a Pool

LUTHOR HD Pool provides exceptional sensitivity and high correlation, even for inputs as low as one cell.

Figure 1 | LUTHOR HD Pool 3’ mRNA-Seq Read Statistics. DU145 cell line, separate pools of 8 single cells, 8x 10 cells, and 8x 100 cells. A) Scatter plot showing average gene number (after UMI collapsing) per cell for 8x 1 cell (dark blue), 8x 10 cells (light green), and 8x 100 cells (light gray) as a function of read depth. Gene detection threshold: ≥1 CPM. B) Same as A, but UMI counts (gene-based collapsed). C) Correlation plots with two exemplary samples for each pool, orthogonal R2 value computed on 8 samples (28 combinations); downsampled to 1 M read depth at CPM ≥1.

High Gene Detection Homogeneity between Samples


A)
(cells) RSD: 1.6%
86.3	85.7	88.2	86.6	85.4	85.1
87.6	85.5	85.4	82.1	86.6	88.3
82.1	86.6	88.3	86.1	87.2	87.8
87.7	88.8	87.2	86.4	86.2	86.6
86.2	86.1	88.4	88.5	85.8	85.2
84.5	85.5	86.7	87.9	86.2	87.0
85.4	87.3	87.0	86.9	86.4	86.8
87.3	87.2	89.3	88.1	87.6	87.5


(UHRR) RSD: 1.4%
80.6	81.4	80.9	80.8	80.1	80.7	79.9	77.7	80.6	80.0	80.8	81.5
79.9	81.3	81.3	81.1	81.5	80.6	80.8	80.8	80.6	81.3	80.0	79.6
81.0	80.7	80.7	80.2	79.8	80.5	80.8	75.5	80.3	79.1	80.4	81.3
80.5	81.6	80.7	80.6	79.1	81.0	80.7	81.8	81.3	80.6	79.6	79.6
80.0	80.7	81.0	79.6	80.8	79.2	80.9	78.0	81.4	81.0	80.6	81.0
82.3	80.4	80.9	77.0	80.5	81.4	82.2	80.9	80.1	81.7	80.3	81.2
80.9	80.7	80.6	81.1	77.3	81.5	81.6	80.2	79.7	80.0	80.0	79.6
79.9	82.0	80.3	80.8	79.8	80.5	81.8	79.1	79.9	78.3	78.7	80.9


B)
(cells) RSD: 6.7%
11’160	10’180	10’447	10’149	10’393	10’503
10’880	9’970	9’878	7’697	11’257	10’942
9’437	11’178	10’095	11’000	10’910	10’807
10’793	9’607	11’016	9’758	10’225	10’550
10’667	11’116	9’275	9’661	10’391	10’012
11’676	10’557	10’967	11’438	10’126	10’073
10’311	10’196	11’039	10’526	10’225	9’465
10’515	10’531	10’644	11’934	10’523	10’446


(UHRR) RSD: 3.2%
11956	12290	11935	12110	12197	12185	11639	10894	12123	11157	11963	12265
10976	12180	11932	12400	12461	12101	11724	11769	12219	12207	11885	10923
12495	12726	12152	11899	11925	11943	12531	12104	12281	12464	12370	12426
12090	12565	12283	12065	11839	11758	11987	12578	12016	12090	11974	12225
11604	12133	12103	12004	11818	11873	12178	11392	12158	12057	11896	12233
12816	11787	12238	11696	12403	12353	12171	11827	11552	12136	11991	12356
12134	11977	12463	12358	11681	12359	12849	12251	11902	10541	11750	11679
11918	11851	12390	12175	11995	11889	11827	11888	11972	11588	11652	12136

Table 1 | Series of 48 (cells) and 96 (UHRR) Pooled Libraries Prepared with LUTHOR HD Pool. Single DU 145 cells and 10 pg Universal Human Reference RNA input (UHRR, Agilent Cat No 740000). A) exonic read percentage (vs. intronic/intergenic, unique, and multimapped reads). B) number of genes. For B, data was downsampled (gene-based collapsing) to 1M reads (cells) and 1M*, 0.5M**, 0.25M** reads (UHRR). RSD: Relative Standard Deviation. * samples in bold italic received less than 1M reads during sequencing. ** data not shown (patterns are very similar to 1M)

Minimal Cross-contamination

Pooled ngs library preparation approaches are inherently prone to some level of contamination between samples. At Lexogen, we made sure that cross-contamination is reduced to almost undetectable levels. Even when tips are not changed!


A)
	UHRR	Arabidopsis thaliana	Cross-contamination
UHRR (6 samples)	3,096,073	649	0.0%
Arabidopsis thaliana (2 samples)	3,299	1,128,368	0.3%
			total	0.1%
B)
	UHRR	Arabidopsis thaliana	Cross-contamination
UHRR (6 samples)	3,245,024	595	0.0%
Arabidopsis thaliana (2 samples)	3,208	1,175,775	0.3%
			total	0.1%

Table 2 | Assessment of Cross-contamination between Pooled Samples. 2 samples of Arabidopsis thaliana RNA (200 pg each) and 6 samples of UHRR (200 pg each) were pooled and prepared with LUTHOR HD Pool. Gene counts measured on data downsampled to 1M read/sample (read counts are cumulated in the table). Cross-contamination calculated as the percentage of cross-species gene counts over total species-specific gene counts.
A) Tips are changed between each sample. B) Tips are not changed between samples during THOR reaction.

Step1:

Total time for Step:

Hands-on time for Step :

Single animal cells (e.g., sorted by FACS) or ultra-low input RNA (10 pg - 10 ng)

The primer contains Unique Molecular Identifiers (UMI, in red), a sample barcode (LPi1, in light blue), a partial Illumina-compatible P7 linker (blue), and a T7 promoter sequence (light brown).

The proprietary end repair step removes the single-stranded 3’ poly(A) overhang and generates a double stranded T7 promoter sequence for RNA amplification.

After the initial THOR reaction, samples are combined by pooling, therefore saving time and effort during the subsequent protocol steps.

During a 2-hour (optional: overnight) in vitro transcription step the original mRNA template is copied repeatedly by linear amplification.

Antisense-RNA copies are generated which contain the partial Illumina compatible P7 linker at the 5’ end.

LUTHOR library generation is initiated by random priming using Lexogen’s proprietary Displacement Stop technology. Random primers contain partial Illumina-compatible P5 sequences (green).

During the library amplification step sequences required for cluster generation are introduced. Optionally, i5 and i7 indices (e.g., Lexogen 12 nt UDI) can be introduced. These optional indices are required for multiplexing more than 96 samples.

LUTHOR HD Pool 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 HD Pool libraries should be sequenced in asymmetric PE mode: Read 1 for the insert, Read 2: 24 nt, i.e., 12 nt for the LPi1 sample barcode and 12 nt for the UMI.

Frequently Asked Questions

Access our frequently asked question (FAQ) resources via the buttons below.

Please also check our General Guidelines and FAQ resources!

How do you like the new online FAQ resource? Please share your feedback with us!

LUTHOR HD Pool Single-Cell 3' mRNA-Seq

User Guide – release 09.07.2024

Product Flyer for High-throughput RNA-Seq for Drug Discovery – release 13.09.2024

i1 12 nt RT-Set for LUTHOR HD Pool – release 13.08.2024

Lexogen UDI 12 nt Unique Dual Index Sequences – update 26.03.2021

Safety Data Sheet

Request Safety Data Sheet

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

Cat. No.	Product Name
205.96	LUTHOR High-Definition Pool Single-Cell 3′ mRNA-Seq Library Prep Kit, 96 preps

LUTHOR HD Pool

High-Definition single-cell RNA sequencing in one tube

Check the FAQs

LUTHOR HD Configurator

LUTHOR HD POOL

Down to single cells or 10 pg RNA input

Down to 2 EUR per library

Up to 36,864 samples per run

Up to 2,548 saved pipet tips (96 preps)

LUTHOR High-Definition Pool Single-Cell 3′ mRNA-Seq Library Prep Kit

Running large scale projects?

RNA-Seq directly from lysates

Performance

Workflow

FAQ

Frequently Asked Questions

Downloads

LUTHOR HD Pool Single-Cell 3' mRNA-Seq

Safety Data Sheet

Ordering Information

Cart

Product

Product

Product

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LUTHOR HD Pool

High-Definition single-cell RNA sequencing in one tube

Check the FAQs

LUTHOR HD Configurator

LUTHOR HD POOL

Down to single cells or 10 pg RNA input

Down to 2 EUR per library

Up to 36,864 samples per run

Up to 2,548 saved pipet tips (96 preps)

LUTHOR High-Definition Pool Single-Cell 3′ mRNA-Seq Library Prep Kit

Running large scale projects?

RNA-Seq directly from lysates

Performance

Workflow

FAQ

Frequently Asked Questions

Downloads

LUTHOR HD Pool Single-Cell 3' mRNA-Seq

Safety Data Sheet

Ordering Information

Cart

Product

Product

Product

My Account

Login

Register