This kit is a total RNA library preparation kit for the Illumina and MGI sequencing platform, including RNA fragmentation reagents, reverse transcription reagents, conventional and strand-specific ds-cDNA synthesis reagents, and library amplification reagents. The sequencing library can be constructed followed by the mRNA purification kit(Cat#12629) or rRNA removal kit(Cat#122257 or Cat#12254). The two-strand synthesis module is equipped with two buffers to meet the need for routine library or strand-specific library. Among them, dTTP is replaced with dUTP in the strand-specific two-strand synthesis Buffer, so dUTP can be added to the second strand of cDNA. The high-fidelity DNA polymerase used in this kit cannot amplify the DNA template containing uracil, achieving strand specificity. All reagents provided have undergone strict quality control and functional verification, ensuring the stability and reproducibility of library preparation to the greatest extent.

Features

Simplified process: two-strand synthesis, end repair and A addition are combined into one step.

Wide compatibility: Compatible with strand-specific and non-strand-specific libraries: dNTP or dUTP amplification is used to achieve non-strand-specific or strand-specific library preparation. And compatible with Illumina and MGI libraries: primer mix for illumina or primer mix for MGI is used to achieve dual-platform compatibility.

High-quality library preparation: Excellent sequencing data quality makes the coverage of transcript regions more uniform.

Specifications

Components

Components No.		Name	12308ES08	12308ES24	12308ES96	12308ES98
12308-A		2× Frag/Prime Buffer	80 μL	250 μL	930 μL	9.7 mL
12308-B		1st Strand Enzyme Mix	16 μL	48 μL	192 μL	2 mL
12308-C		Strand Specificity Reagent	50 μL	150 μL	580 μL	6.042 mL
12308-D		2nd Strand Buffer (dNTP)	240 μL	720 μL	2×1440 μL	30 mL
12308-E		2nd Strand Buffer (dUTP)	240 μL	720 μL	2×1440 μL	30 mL
12308-F		2nd Strand Enzyme Master Mix	40 μL	120 μL	480 μL	5 mL
12308-G		Ligation Enhancer	240 μL	720 μL	2×1440 μL	30 mL
12308-H		Novel T4 DNA Ligase	40 μL	120 μL	480 μL	5 mL
12308-I		2×Super Canace™ II High-Fidelity Mix	200 μL	600 μL	2×1200 μL	25 mL
12308-K		Nuclease Free H2O	100 μL	300 μL	1000 μL	1000 mL
12308-*	*	Primer mix	NA	NA	NA	NA
[Note]*: Primer mix is a necessary reagent for the library with completed adapter(short adapter not required), but it is not included in this kit and needs to be configured separately. The components of this kit are compatible with both Illumina and MGI platforms, but additional primer mixes specific to Illumina or MGI(Cat# 13335 Primer Mix for Illumina and Cat# 13334 Primer Mix for MGI) are required.

Workflow

Application

RNA library preparation; LncRNA library preparation; M6A library preparation; transcriptome analysis; gene expression analysis; alternative splicing analysis; fusion gene detection;

Figures

1. RNA fragmentation condition selection

By optimizing the fragmentation system, the mRNA fragmentation effect of this kit is more concentrated and the effect is better. The fragmentation conditions are as follows:

94℃ 5min: 400~500bp;

94℃ 7min: 350~400bp;

94℃ 10min: 250~300bp.

Figure 1. Fragment size analysis under different RNA fragmentation conditions

2. Low-quality samples, high library preparation success rate

Figure 2. 500 ng input of different DV200 Human FFPE RNA libraries.

*DV200 indicates the proportion of RNA fragments larger than 200 nt in the sample. For severely degraded FFPE samples, the DV200 value can better reflect the quality of the sample.

Conclusion:

Compared with other suppliers N*, yeasen RNA library preparation kit: High library preparation yield, high data utilization, low rRNA residual rate, and high exon ratio.

3. Chain-specific library preparation, high specificity

4. High consistency of multi-sample sequencing data

Figure 3. Consistency of library preparation and sequencing of samples with different starting amounts (samples are human RNA samples)

5. Stability test

Figure 4. Freeze-thaw stability(a) and room temperature stability testing(b)