Hieff NGS® DNA&RNA Library Co-Prep Kit V2 is a DNA&RNA co-library kit for Illumina®&MGI® sequencing platform, which contains efficient cDNA synthesis reagent and enzyme digestion reagent. Compared with the traditional library construction method, this product can efficiently complete cDNA synthesis and DNA&RNA library construction in one tube.The kit contains high quality enzyme for DNA fragmentation and combines DNA fragmentation, end-repair, and dA-tailing into one step, which reduce time significantly and cost of library preparation. This library prep kit has an excellent library conversion rate and is applicable for samples from all common animals, plants, microorganisms, etc., and also the FFPE samples. This upgraded kit using the latest optimized ligase greatly decreases the self-ligation rate during adapter ligation. Moreover, the introduction of a new high-fidelity polymerase further improves the homogeneity and fidelity of amplification.

All the components provided by the kit have undergone strict quality control and functional verification, which ensures the stability and repeatability of library construction to the greatest extent.

Specifications

Cat.No.	12305ES08 / 12305ES24 / 12305ES96
Size	8 T / 24 T / 96 T

Components

No.		Name	12305ES08	12305ES24	12305ES96
12305-A		Random Primer	20 μL	60 μL	240 μL
12305-B		cDNA Reaction Buffer	64 μL	192 μL	768 μL
12305-C		cDNA Enzyme Mix	16 μL	48 μL	192 μL
12305-D		Smearase Buffer	80 μL	240 μL	960 μL
12305-E		Smearase Enzyme Mix	40 μL	120 μL	480 μL
12305-F		Ligation Enhancer	240 μL	720 μL	2×1440 μL
12305-G		Novel T4 DNA Ligase	40 μL	120 μL	480 μL
12305-H		2×Super Canace® II High-Fidelity Mix	200 μL	600 μL	2×1200 μL
*		Primer Mix*	40 μL	120 μL	480 μL

Note: * indicates that this reagent is not included in this kit and additional reagents are required.The kit is compatible with dual platforms of Illumina^®&MGI^®, but additional primer mix (CAT # 13334 Primer Mix for MGI^® and Cat# 13335 Primer Mix for Illumina^®) is required.

Work flow:

Storage

This product should be stored at -25~-15℃ for 1 year.

Notes

About the operation

1. 1. Please operate with lab coats and disposable gloves，for your safety.
2. Thaw components at room temperature.After thawing, mix thoroughly by vortexing, spin the tube briefly and place them on ice for later use.
3. It is recommended to perform each reaction step in a thermocycler with a heated lid. The thermocycler should be preheated to the set temperature before use.
4. Please use consumables without RNasecontamination., andcleaning the experimental area regularly. ThermoFisher's RNAZap^TM high-efficiency nucleic acid removal spray was recommended to remove RNase contamination.
5. Improper operations may very likely cause aerosol contaminations, impacting the accuracy of result. Mandatory physical isolation of PCR reaction mixing regions and PCR product purification assay regions is recommended. Equipped with equipment such as specialized pipettes for library construction.
6. 6. This product is for research use only.

Adapter Ligation

1. Illumina or MGI Long Adapter (Barcoded Adapter) kits and short Adapter kits are available for customers to choose according to their experimental requirements.
2. Selecting high-quality, commercial adapters was recommended. If self-made adapters are selected, please entrust a company with experience in NGS primer synthesis and remark the need for strict contamination control. In addition, it is recommended to prepare DNA annealing solution in a clean bench and only operate one type of adapter each time to prevent cross-contamination.
3. Please thaw the adapters on the ice or at 4°C; when operating at room temperature, the laboratory temperature should not exceed 25°C to prevent the adapters from denaturing.
4. The concentration of the adapter directly affects the ligation efficiency and library yield. The adapter volume added to the kit is fixed to 5 μl. The adapters are recommended to be diluted with 0.1×TE buffer and the diluted adapters can be stored at 4°C for 48 hours. Table1 lists the recommended adapter amount for different amounts of input RNA.

Table 1-1 The recommended Illumina^® adapter amount for different input DNA&RNA

Input Total DNA&RNA	Illumina® Adapter stock concentration
<10 ng	3 μM
≥10 ng	15 μM

 

Table 1-2 The recommended MGI^® adapter amount for different input DNA&RNA

Input Total DNA&RNA	MGI® Adapter stock concentration
<10 ng	5 μM
≥10 ng	10 μM

*The Adapter usage can be adjusted according to different types of Total RNA samples and input amount.

Library Amplification

Amplification cycle numbers should be strictly controlled. Insufficient amplification may lead to low library yield; Over-amplification may introduce increased bias, errors, duplicated read, and chimeric products. Table 2 lists recommended cycle numbers targeting the library yield of 1 μg.

Table 2 The recommended number of cycles to generate DNA&RNA library *

Input Total DNA&RNA	Number of cycles
<1 ng	10~12
1 ng	9~10
10 ng	6~7
50 ng	4~5
100~1000 ng	4

Note：*The yield of the library is not only related to the input quantity and the number of amplification cycles, but also affected by the quality of samples, fragmentation conditions and sorting conditions. In the process of library construction, choose the most appropriate conditions according to the actual situation.

Bead-based DNA Cleanup and Size Selection

1. 1.There are multiple steps in the library construction process that require DNA purification magnetic beads. We recommend Hieff NGS™ DNA Selection Beads (Yeasen Cat#12601) or AMPure® XP magnetic beads (Beckman Cat#A63880) for DNA purification and size-selection.
2. 2.The magnetic beads should be equilibrated at room temperature prior to use, otherwise the yield will decrease and the size selecting effect will be affected.
3. 3.The magnetic beads should be mixed well by vortex or pipetting prior to use.
4. 4.Do not aspirate the beads when transferring the supernatant, even trace amounts of the beads may impact the following reactions.
5. 5.The 80% ethanol should be freshly prepared, otherwise it will affect the recovery efficiency.
6. 6.The magnetic beads should be dried at room temperature before eluting the product. Insufficient dryness will easily cause ethanol residual to affect subsequent reactions; excessive dryness will cause the magnetic beads to crack and reduce the purification yield. Normally, drying at room temperature for 3-5 minutes is enough to allow the beads to fully dry.
7. 7.If needed, the purified or size-selected DNA samples eluted in1× TE buffer can be stored at 4°C for 1-2 weeks or at -20°C for a month.

Library Quality Analysis

1. The constructed libraries quality is generally analyzed by measuring the concentrations and size distributions.
2. Libraries` concentrations can be measured by fluorescent-based methods such as Qubit and PicoGreen or qPCR.
3. It is NOT recommended to use absorbance-based quantification methods such as NanoDrop.
4. It is recommended to use qPCR method for library quantification: fluorescent-based methods such as Qubit and PicoGreen cannot differentiate the incomplete dsDNA structures (inserts with no adapter or with only one of the ends ligated with adapter) from the complete libraries. The qPCR method will only amplify and measure the complete libraries with both ends ligated with adapters (the sequencable libraries), thus providing a more accurate measurement for loading.
5. The size distribution of libraries can be analyzed using Agilent Bioanalyzer or other devices based on the principles of capillary electrophoresis or microfluidics.

Other Materials

1. 1.DNA purification magnetic beads: Hieff NGS™ DNA Selection Beads (Yeasen Cat#12601) or AMPure^®XP Beads (A63880) or other equivalent products.

2.Adapters: Complete Adapter for Illumina (Yeasen Cat#13519-13520 or other equivalent products) or Complete Adapter for MGI (Yeasen Cat#13360-13362 or other equivalent products).

3. Library quality analysis: Agilent 2100 Bioanalyzer DNA 1000 Chip/ High Sensitivity Chip or other equivalent products; library quantitative reagents.
4. Other materials: absolute ethanol, sterile ultrapure water, low retention pipette tips, PCR tube, magnetic stands, thermal cycler, etc.

 

Documents:

Manuals

12305ES-Hieff NGS® DNA&RNA Library Co-Prep Kit V2.pdf