NadPrep EZ RNA Library Preparation Kit

Feature Highlight

Broad Coverage

● Supports 2.5-500 ng initial input
● Compatible with various sample types and qualities
● Flexible choice of non-stranded or stranded library preparation
● Adapters compatible with mainstream NGS platforms
● Seamless integration with upstream and downstream modules for diverse applications

Superior Performance

● Higher library yield
● More uniform transcript coverage
● Higher detection sensitivity for low-abundance transcripts and fusion genes
● Higher strand specificity
● Excellent library complexity

Fast Workflow

● Combines 2nd Strand cDNA Synthesis, End Repair & A-tailing into a single step

● Greatly streamlines the workflow and user-friendly operation, significantly shortens library preparation duration

Workflow

Performance

Stable and Efficient Library Yield

I. Compatible with a Wide Range of Input Amounts

Figure 1. Library yield for cell line RNA samples with different input amounts. The non-stranded libraries were prepared with RNA samples across different input amounts using the NadPrep EZ RNA Library Preparation Module coupled with the NadPrep Universal Stubby Adapter (UDI) Module (NovaSeq) and NadPrep Universal Adapter (MDI) Module (for MGI) (DNBSEQ) respectively, following the recommended PCR cycles as described in the user manuals. A. Total RNA [connected with the downstream workflow (RNACap)]; B. rRNA-depleted [connected with the upstream workflow (rRNA depletion): NadPrep rRNA Blocking Reagent (Human)].
Note: All the initial samples are Human Brain Total RNA (Takara, 636530).

II. Compatible with Multiple Types Samples of Different Grades

Figure 2. Library yield for RNA samples with different grades. The non-stranded libraries were prepared with RNA samples with different grades. A. Total RNA; B. rRNA-depleted.
Note: Sample grading standard: Grade A (cell line RNA): Human Brain Total RNA (Takara, 636530), RIN ≥ 7; Grade C (FFPE-derived RNA): DV₂₀₀ ≥ 50; Grade E (FFPE-derived RNA): DV₂₀₀ < 50. All the initial input amounts were 50 ng.

III. Higher library yield

Figure 3. Nadprep EZ RNA Library Preparation Kit has a more efficient library yield than Vendor A & B. The non-stranded libraries were prepared with RNA samples across different grades using the NadPrep EZ RNA Library Preparation Module (NadPrep), Vendor A and Vendor B, coupled with the NadPrep Universal Stubby Adapter (UDI) Module respectively. A. Total RNA; B. rRNA-depleted.

More Uniform Transcript Coverage

Figure 4. NadPrep EZ RNA Library Preparation Kit performs more uniform transcript coverage compared to Vendor A & B. The non-stranded libraries were prepared with RNA samples using NadPrep, Vendor A, and Vendor B coupled with the NadPrep Universal Stubby Adapter (UDI) Module respectively, connected with the upstream workflows A.rRNA-depleted, B. enriched mRNA; and the downstream workflow C. RNACap, with 500 ng of pre-library input for hybridization capture using the NanOnco Plus Panel v2.0 and NadPrep Hybrid Capture Reagents to asses transcript coverage. Sequencing was performed on NovaSeq 6000, PE150. For each sample, 0.3 Gb was randomly selected for data analysis.
Note: The samples in figure B are cell line RNA, with an initial input of 50 ng both in figure A & B. The samples in figure C are cell line mRNA, with an initial input of 10 ng.

Higher Detection Sensitivity

Figure 5. NadPrep EZ RNA Library Preparation Kit performs higher sensitivity for low-abundance transcripts compared to Vendor A & B. The non-stranded pre-libraries were prepared using NadPrep, Vendor A, and Vendor B coupled with the NadPrep Universal Stubby Adapter (UDI) Module respectively. 500 ng of each pre-library was performed for hybridization capture using the Ext-RNA Control Panel v1.0 and NadPrep Hybrid Capture Reagents. A. No. of ERCC detected; B. ERCC RPKM.
Note: The samples are cell line RNA spiked with 1 μL ERCC (1:1,000), with an initial input of 50 ng.

Figure 6. NadPrep EZ RNA Library Preparation Kit performs higher detection sensitivity for fusion gene than Vendor A & B in standards. The non-stranded libraries were prepared using NadPrep, Vendor A, and Vendor B coupled with the NadPrep Universal Stubby Adapter (UDI) Module respectively. 500 ng of each pre-library was performed for hybridization capture using the OncoFu Elite (for RNA) Panel v1.0 and NadPrep Hybrid Capture Reagents.
Note: The samples are mixture of FFPE RNA (Grade C) mixed with 3.125% Onco Fusion Multiplex FFPE(DNA/RNA) Reference Standard (GeneWell, GW-RPSM1006)], with an initial input of 100 ng.

Higher Strand Specificity

Figure 7. The NadPrep Rapid RNA Library Preparation Kit performs high strand specificity across RNA of different grades. The strand specificity percentage of stranded libraries were prepared with RNA samples using NadPrep, Vendor B, and Vendor C combined with the NadPrep Universal Stubby Adapter (UDI) Module respectively, following A. Upstream rRNA rapid blocking module, B. mRNA enrichment, C. Downstream RNACap (500 ng pre-library hybridized with the NanOnco Plus Panel v2.0 and the NadPrep Hybrid Capture Reagents). Sequencing platform: NovaSeq 6000, PE150. For each sample, 0.3 Gb of data was randomly selected for data analysis.
Note: The samples in figure A are Grade A (cell line RNA), with an initial input of 50 ng both in Figure A & C. The samples in figure B are cell line mRNA, with an initial input of 5 ng.

Seamless Connection Upstream and Downstream Modules, Broad Application Compatibility

I. Connection with RNACap

Figure 8. Performance of the stranded libraries prepared with the downstream workflow (RNACap). The stranded libraries were prepared with RNA samples across different input amounts using the NadPrep EZ RNA Library Preparation Module coupled with the NadPrep Universal Adapter (MDI) Module (for MGI) (DNBSEQ). 500 ng of each pre-library was performed for hybridization capture using the NanOnco Plus Panel v2.0 and NadPrep Hybrid Capture Reagents. A. rRNA ratio; B. No. of genes detected. Sequencing was performed on DNBSEQ-T7, PE150. For each sample, 0.3 Gb of data was randomly selected for data analysis.

II. Connection with Upstream rRNA Rapid Blocking

Figure 9. Library preparation performance following upstream rRNA rapid blocking. A. rRNA ratio; B. No. of genes detected.

For research use only. Not for use in diagnostic procedures.

Library Prep Module

Package	Color of Cap Tube	Component	Volume 1002441 (24 rxn)	Volume 1002442 (96 rxn)
Box1		Random Primer II	30 µL	115 µL
		1^st Strand Buﬀer	120 µL	460 µL
		1^st Strand Buﬀer (stranded)	120 µL	460 µL
		1^st Strand Enzyme	60 µL	230 µL
		2^nd Strand & EA Buﬀer	750 µL	2×1500 µL
		2^nd Strand & EA Buﬀer (with dUTP)	750 µL	2×1500 µL
		2^nd Strand & EA Enzyme	120 µL	460 µL
		Ligation Buffer	750 µL	2×1500 µL
		DNA Ligase	65 µL	250 µL
		2X HiFi PCR Master Pro Mix	720 µL	2×1600 µL
		TE Solution	1 mL	4 mL
	/	Nuclease Free Water	2.5 mL	8 mL
Box2	/	NadPrep SP Beads	4 mL	15 mL

Universal Adapter (SI) Module (for MGI)

Color of Cap Tube	Component	Volume 1003611 (24 rxn)	Volume 1003621 (96 rxn)	Volume 1003622(96 rxn)
	NadPrep M-Index (SI) Primer Mix #	12×15 µL (Tube)	24×25 µL (Tube)	24×25 µL (Tube)
	NadPrep M-Adapter (SI)	60 µL (Tube)	230 µL (Tube)	230 µL (Tube)

Color of Cap Tube	Component	Volume 1003641 (96 rxn)
Color of Cap Tube	Component	Volume 1003641 (96 rxn)
	NadPrep M-Index (SI) Primer Mix #	96×8 µL (Plate)
	NadPrep M-Adapter (SI)	260 µL (Tube)
	NadPrep M-Amplification Primer Mix (for MGI, SI)	75 µL (Tube)

Universal Adapter (MDI) Module (for MGI)

Color of Cap Tube	Component	Volume 1003711 (24 rxn)	Volume 1003721 (96 rxn)	Volume 1003722 (96 rxn)	Volume 1003725 (96 rxn)	Volume 1003726 (96 rxn)
	NadPrep M-Index (MDI) Primer Mix #	12×15 µL (Tube)	24×25 µL (Tube)	24×25 µL (Tube)	24×25 µL (Tube)	24×25 µL (Tube)
	NadPrep M-Adapter (DI)	60 µL (Tube)	230 µL (Tube)	230 µL (Tube)	230 µL (Tube)	230 µL (Tube)

Color of Cap Tube	Component	Volume 1003751 (96 rxn)	Volume 1003752 (96 rxn)	Volume 1003753 (96 rxn)	Volume 1003754 (96 rxn)
Color of Cap Tube	Component	Volume 1003751 (96 rxn)	Volume 1003752 (96 rxn)	Volume 1003753 (96 rxn)	Volume 1003754 (96 rxn)
	NadPrep M-Index (MDI) Primer Mix #	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)
	NadPrep M-Adapter (DI)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)
	NadPrep M-Amplification Primer Mix (for MGI, DI)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)

Color of Cap Tube	Component	Volume 1003761 (1152 rxn)	Volume 1003762 (1152 rxn)	Volume 1003763 (1152 rxn)	Volume 1003764 (1152 rxn)
Color of Cap Tube	Component	Volume 1003761 (1152 rxn)	Volume 1003762 (1152 rxn)	Volume 1003763 (1152 rxn)	Volume 1003764 (1152 rxn)
	NadPrep M-Index (MDI) Primer Mix #	96×72 µL (Tube)	96×72 µL (Tube)	96×72 µL (Tube)	96×72 µL (Tube)
	NadPrep M-Adapter (DI)	2×1350 µL (Tube)	2×1350 µL (Tube)	2×1350 µL (Tube)	2×1350 µL (Tube)
	NadPrep M-Amplification Primer Mix (for MGI, DI)	900 µL (Tube)	900 µL (Tube)	900 µL (Tube)	900 µL (Tube)

Universal Stubby Adapter (UDI) Module（for Illumina）

Color of Cap Tube	Component	Volume 1003240 (24 rxn)	Volume 1003241 (96 rxn)	Volume 1003242 (96 rxn)
	NadPrep Universal UDI-index Primer Mix #	12×15 µL (Tube)	24×25 µL (Tube)	24×25 µL (Tube)
	NadPrep Universal Stubby Adapter	60 µL (Tube)	230 µL (Tube)	230 µL (Tube)
	NadPrep Amplification Primer Mix II	20 µL (Tube)	75 µL (Tube)	75 µL (Tube)

Color of Cap Tube	Component	Volume 1003251 (96 rxn)	Volume 1003252 (96 rxn)	Volume 1003253 (96 rxn)	Volume 1003254 (96 rxn)	Volume 1003255 (96 rxn)	Volume 1003256 (96 rxn)	Volume 1003257 (96 rxn)	Volume 1003258 (96 rxn)
Color of Cap Tube	Component	Volume 1003251 (96 rxn)	Volume 1003252 (96 rxn)	Volume 1003253 (96 rxn)	Volume 1003254 (96 rxn)	Volume 1003255 (96 rxn)	Volume 1003256 (96 rxn)	Volume 1003257 (96 rxn)	Volume 1003258 (96 rxn)
	NadPrep Universal UDI-index Primer Mix #	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)	96×8 µL (Plate)
	NadPrep Universal Stubby Adapter	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)	260 µL (Tube)
	NadPrep Amplification Primer Mix II	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)	75 µL (Tube)

Color of Cap Tube	Component	Volume 1003261 (1152 rxn)	Volume 1003262 (1152 rxn)	Volume 1003263 (1152 rxn)	Volume 1003264 (1152 rxn)
Color of Cap Tube	Component	Volume 1003261 (1152 rxn)	Volume 1003262 (1152 rxn)	Volume 1003263 (1152 rxn)	Volume 1003264 (1152 rxn)
	NadPrep Universal UDI-index Primer Mix #	96×72 µL (Tube)	96×72 µL (Tube)	96×72 µL (Tube)	96×72 µL (Tube)
	NadPrep Universal Stubby Adapter	2×1350 µL (Tube)	2×1350 µL (Tube)	2×1350 µL (Tube)	2×1350 µL (Tube)
	NadPrep Amplification Primer Mix II	900 µL (Tube)	900 µL (Tube)	900 µL (Tube)	900 µL (Tube)

For research use only. Not for use in diagnostic procedures.

What can cause low library yield when using the NadPrep EZ RNA Library Preparation Kit? How to solve it?

RNase contamination: Use RNase-free consumables throughout the workflow and perform all the operations in RNase-free conditions. RNase contamination will reduce reverse transcription efficiency and low library yield.
Low sample purity: Residual contaminants in RNA (e.g. chelators, guanidine salts, phenol, ethanol) may inhibit the activity of reverse transcriptase. It is recommended to purify RNA according to Step 7: Post-amplification Cleanup in the use manual using 2X NadPrep SP Beads and elute in Nuclease Free Water.
Poor sample quality: For low-quality RNA samples, follow the user manual’s recommendations to increase the no. of PCR cycles appropriately.

Is the NadPrep EZ RNA Library Preparation Kit suitable for severely degraded FFPE samples?

Yes. It is. NadPrep EZ RNA Library Preparation Kit is suitable to most RNA samples from different grades and sources (cells, fresh tissues etc.). For FFPE-derived RNA samples with severe fragmentation (DV₂₀₀ < 20), reverse transcription efficiency may be reduced, and it is recommended to increase the no. of PCR cycles as appropriate. When performed targeted capture on FFPE-derived RNA samples, consider increasing the RNA input to improve capture efficiency.

Which sample types are the NadPrep EZ RNA Library Preparation Kit applicable for?

NadPrep EZ RNA Library Preparation Kit is widely suitable for the libraries prepared from total RNA with different grades, poly(A)-enriched mRNA, and rRNA-depleted RNA. It should be noted that the mRNA content in total RNA from different sources varies greatly. If the initial input amount of total RNA is too low, sufficient mRNA may not be obtained to prepare a high-quality library.

What’s the difference between non-stranded and stranded libraries from NadPrep EZ RNA Library Preparation Kit?

RNA-Seq library preparation generally falls into two types: non-stranded (general) and stranded (strand-specific). The core difference is whether the library retains transcript directional information. Among them, stranded libraries retain the transcript directional information during the preparing process, and sequencing and downstream analysis can determine whether the transcripts originate from the sense or antisense strands of DNA. However, non-stranded libraries lose directional information during cDNA synthesis, and sequencing data cannot directly indicate transcript directions. Compared with general RNA-Seq, strand-specific RNA-Seq provides more accurate transcript quantification, clearer gene structures, identification of antisense transcripts, and discovery of novel transcripts. Therefore, stranded RNA-Seq is crucial for studies of gene structure and expression regulation. To obtain transcript direction information, stranded libraries should be preferred.

Type	Product	Detail	Catalog#
Library Prep Module	NadPrep EZ RNA Library Preparation Module, 24 rxn	24 rxn	1002441
Library Prep Module	NadPrep EZ RNA Library Preparation Module, 96 rxn	96 rxn	1002442
Universal Adapter (SI) Module (for MGI)	NadPrep Universal Adapter (SI) Module Set A1 (for MGI), 24 rxn	SI 1-12	1003611
	NadPrep Universal Adapter (SI) Module Set E1 (for MGI), 96 rxn	SI 1-96	1003641
	NadPrep Universal Adapter (SI) Module Set B1 (for MGI), 96 rxn	SI 1-24	1003621
	NadPrep Universal Adapter (SI) Module Set B2 (for MGI), 96 rxn	SI 25-48	1003622
Universal Adapter (MDI)Module (for MGI)	NadPrep Universal Adapter (MDI) Module Set A1 (for MGI), 24 rxn	MDI 1-12	1003711
	NadPrep Universal Adapter (MDI) Module Set B1 (for MGI), 96 rxn	MDI 1-24	1003721
	NadPrep Universal Adapter (MDI) Module Set B2 (for MGI), 96 rxn	MDI 25-48	1003722
	NadPrep Universal Adapter (MDI) Module Set B3 (for MGI), 96 rxn	MDI 49-72	1003725
	NadPrep Universal Adapter (MDI) Module Set B4 (for MGI), 96 rxn	MDI 73-96	1003726
	NadPrep Universal Adapter (MDI) Module Set E1 (for MGI), 96 rxn	MDI 1-96	1003751
	NadPrep Universal Adapter (MDI) Module Set E2 (for MGI), 96 rxn	MDI 97-192	1003752
	NadPrep Universal Adapter (MDI) Module Set E3 (for MGI), 96 rxn	MDI 193-288	1003753
	NadPrep Universal Adapter (MDI) Module Set E4 (for MGI), 96 rxn	MDI 289-384	1003754
	NadPrep Universal Adapter (MDI) Module Set F1 (for MGI), 1152 rxn	MDI 1-96	1003761
	NadPrep Universal Adapter (MDI) Module Set F2 (for MGI), 1152 rxn	MDI 97-192	1003762
	NadPrep Universal Adapter (MDI) Module Set F3 (for MGI), 1152 rxn	MDI 193-288	1003763
	NadPrep Universal Adapter (MDI) Module Set F4 (for MGI), 1152 rxn	MDI 289-384	1003764
Universal Stubby Adapter (UDI) Module	NadPrep Universal Stubby Adapter (UDI) Module Set A1, 24 rxn	Index 1-12 24 rxn	1003240
	NadPrep Universal Stubby Adapter (UDI) Module Set B1, 96 rxn	Index 1-24 96 rxn	1003241
	NadPrep Universal Stubby Adapter (UDI) Module Set B2, 96 rxn	Index 25-48 96 rxn	1003242
	NadPrep Universal Stubby Adapter (UDI) Module Set E1, 96 rxn	Index 1-96 96 rxn	1003251
	NadPrep Universal Stubby Adapter (UDI) Module Set E2, 96 rxn	Index 97-192 96 rxn	1003252
	NadPrep Universal Stubby Adapter (UDI) Module Set E3, 96 rxn	Index 193-288 96 rxn	1003253
	NadPrep Universal Stubby Adapter (UDI) Module Set E4, 96 rxn	Index 289-384 96 rxn	1003254
	NadPrep Universal Stubby Adapter (UDI) Module Set E5, 96 rxn	Index 385-480 96 rxn	1003255
	NadPrep Universal Stubby Adapter (UDI) Module Set E6, 96 rxn	Index 481-576 96 rxn	1003256
	NadPrep Universal Stubby Adapter (UDI) Module Set E7, 96 rxn	Index 577-672 96 rxn	1003257
	NadPrep Universal Stubby Adapter (UDI) Module Set E8, 96 rxn	Index 673-768 96 rxn	1003258
	NadPrep Universal Stubby Adapter (UDI) Module Set F1, 1152 rxn	Index 1-96 1152 rxn	1003261
	NadPrep Universal Stubby Adapter (UDI) Module Set F2, 1152 rxn	Index 97-192 1152 rxn	1003262
	NadPrep Universal Stubby Adapter (UDI) Module Set F3, 1152 rxn	Index 193-288 1152 rxn	1003263
	NadPrep Universal Stubby Adapter (UDI) Module Set F4, 1152 rxn	Index 289-384 1152 rxn	1003264