0102030405
NGS Ultima Dual-mode RNA Library Prep Kit HYD213
NGS Ultima Dual-mode RNA Library Prep 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 or rRNA removal kit. 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.
Components
| Components | 24T | 96T |
|---|---|---|
| 2×Frag/Prime Buffer | 250 μL | 930 μL |
| 1st Strand Enzyme Mix | 48 μL | 192 μL |
| Strand Specificity Reagent | 150 μL | 580 μL |
| 2nd Strand Buffer (dNTP) | 720 μL | 2×1440 μL |
| 2nd Strand Buffer (dUTP) | 720 μL | 2×1440 μL |
| 2nd Strand Enzyme Master Mix | 120 μL | 480 μL |
| Ligation Enhancer | 720 μL | 2×1440 μL |
| Novel T4 DNA Ligase | 120 μL | 480 μL |
| 2×Super High-Fidelity Mix | 600 μL | 2×1200 μL |
| Nuclease Free H2O | 300 μL | 1000 μL |
| Primer mix | NA | NA |
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.
Storage Conditions
Store at -25~-15℃ for 1 year.
Notes
1. About Adapter Ligation
1. We recommend the use of high-quality commercial adapters. If customers use homemade adapters, pleaseentrust a company with experience in NGS primer synthesis and note that strict anti-contaminationcontrol isrequired. In addition, when performing adapter annealing operations, please complete it in a clean bench. Onlyoperate one adapter at a time to prevent cross contamination.2. When using adapters, please take out the adapters in advance and thaw them at 4°C or on an ice box; whenoperating at room temperature, the laboratory temperature should not exceed 25°C to prevent the adaptersfrommelting.
3. During the library preparation process, too high or too low adapter concentrations will lead to a lowlibrarypreparation success rate. In the operation plan of this kit, the adapter volume added is fixed at 5 μL. Pleasereferto Table 1 to dilute the adapter according to the initial RNA input. Please choose 0.1×TE buffer as theadapterdiluent. The diluted adapter can be stored at 4°C for 48 h.
Table 1 Recommended Input Total RNA Amount and Illumina Adapter Concentration
| Input Total RNA | Illumina Adapter stock concentration |
|---|---|
| 10 ng | 1 μM |
| 100 ng | 1.5 μM |
| 500 ng | 3 μM |
| ≥1 μg | 5 μM |
Table 2 Recommended Input Total RNA Amount and MGI Adapter Concentration
| Input Total RNA | MGI Adapter stock concentration |
|---|---|
| 100~499 ng | 2 μM |
| 500~4000 ng | 1.5 μM |
2. About Library Amplification
1) The library amplification component in this kit is composed of our company's second-generation high-fidelity DNA polymerase. Based on the first generation, it greatly enhances the uniformity of amplification, and even low-copy genes can be amplified without preference.
2) The library amplification step requires strict control of the number of amplification cycles. Insufficient cycles will lead to low library yield; excessive cycles will lead to increased library preference, increased repetitiveness, increased chimeric products, accumulation of amplified mutations and other adverse consequences. Table2liststhe recommendations for the input total RNA amount and the corresponding amplification cycle number for library amplification using this kit.
3) The recommended cycle number in Table 2 can meet most library preparation needs. If your sample quality is poor (such as severely degraded FFPE samples), the cycle number can be appropriately increased according to the actual situation. Please pay special attention when constructing mRNA libraries. Since the content of mRNA in Total RNA extracted from different species and tissues varies greatly, the number of amplification cycles needstobe appropriately adjusted according to the starting amount of library preparation, species type and sample processing conditions in the experiment.
1) The library amplification component in this kit is composed of our company's second-generation high-fidelity DNA polymerase. Based on the first generation, it greatly enhances the uniformity of amplification, and even low-copy genes can be amplified without preference.
2) The library amplification step requires strict control of the number of amplification cycles. Insufficient cycles will lead to low library yield; excessive cycles will lead to increased library preference, increased repetitiveness, increased chimeric products, accumulation of amplified mutations and other adverse consequences. Table2liststhe recommendations for the input total RNA amount and the corresponding amplification cycle number for library amplification using this kit.
3) The recommended cycle number in Table 2 can meet most library preparation needs. If your sample quality is poor (such as severely degraded FFPE samples), the cycle number can be appropriately increased according to the actual situation. Please pay special attention when constructing mRNA libraries. Since the content of mRNA in Total RNA extracted from different species and tissues varies greatly, the number of amplification cycles needstobe appropriately adjusted according to the starting amount of library preparation, species type and sample processing conditions in the experiment.
Table 2 Recommended Input Total RNA Amount and Amplification Cycle Number*
| Input Total RNA | Number of cycles | |
|---|---|---|
| Non-stranded | Stranded | |
| 10 ng | 15 | 15 |
| 100 ng | 14 | 14 |
| 500 ng | 12 | 13 |
| ≥1 μg | 11 | 12 |
Since the library yield is not only related to the input amount and the number of amplification cycles, but also sample quality, fragmentation conditions, sorting conditions, etc. will affect the yield. During the library preparation process, please consider theactual situation and choose the most appropriate library preparation conditions
3. Bead-based DNA Cleanup and Size Selection
1) There are multiple steps in the library construction process that require DNA purification magnetic beads.
2) The magnetic bead should be balanced to room temperature before use, otherwise the yield will decrease and the size selecting effect will be affected.
3) The magnetic beads should be mixed well by vortex or pipetting prior to use.
4) Do not aspirate the beads when transferring the supernatant, even trace amounts of the beads may impact the following reactions.
5) The 80% ethanol should be freshly prepared, otherwise it will affect the recovery efficiency.
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) If needed, the purified or size-selected DNA samples eluted in TE buffer can be stored at 4°C for 1-2 weeks or at-20°C for a month.
5. Library Quality Analysis
1. Normally, the quality of the constructed library can be evaluated by length distribution detection and concentration detection.
2. Library concentration detection can be performed using: methods based on double-stranded DNA fluorescent dyes, such as Qubit®, PicoGreen®, etc.; methods based on qPCR absolute quantification.
3. It is recommended to use the qPCR method for library concentration detection: When using concentration determination methods based on double-stranded DNA fluorescent dyes such as Qubit®, it is impossible to effectively distinguish products with single-end connected to Adapter, products without Adapter connected to both ends, and other incomplete double-stranded structure products; qPCR absolute quantification is based on the principle of PCR amplification, and only quantifies libraries with complete Adapters at both ends in the sample(i.e., libraries that can be sequenced), which can exclude the interference of non-sequenceable libraries that are not connected to Adapters at either one end or both ends.
4. Library concentration detection cannot be performed using spectral detection methods, such as NanoDrop®.
5. Library length distribution detection can be performed using devices based on capillary electrophoresis or microfluidics, such as Agilent Bioanalyzer 2100
6. Other Material
1. mRNA enrichment Kit
2. rRNA depletion Kit
3. RNA Cleaner
4. DNA Cleaner
5. RNA quality control
6. Adapters: Complete Adapter for Illumina TM or Complete Adapter for MGITM
7. Library quality control: Agilent 2100 Bioanalyzer DNA 1000 Chip/ High Sensitivity Chip or other equivalent products.
8. Other materials: ethanol, Sterilized ddH2O, PCR tube, magnetic stand, thermal cycler etc.