Fast RNA-seq Lib Prep Kit V2 (Ilumina+MGI) HYD214
Fast RNA-seq Lib Prep Kit V2 (Ilumina+MGI) is an efficient, fast and highly compatible RNA library construction kit. It is suitable for Total RNA samples of eukaryotic organisms such as animals, plants, fungi, or purified mRNA samples; 10-1000 ng of RNA samples can be constructed into sequencing libraries suitable for MGI and Illumina high-throughput sequencing platforms. This kit is compatible with both Full Adapter and Truncated Adapter of MGI and Illumina sequencing platforms. This kit combines the RNA library construction process Second strand cDNA synthesis, End repair and dA-tailing into one step, shortening the total time from 7 hr to 3 hr, greatly shortening the library construction time; at the same time, the kit provides two types of Second Strand & dA Buffer, and customers can choose the corresponding Second Strand & dA Buffer or Second Strand & dA Buffer with dUTP for non-strand-specific library construction or strand-specific library construction according to their needs. Each reagent in the kit has undergone strict quality control, and each batch of library construction Kit has been verified by library construction and sequencing, ensuring the stability of the performance of each batch of kits.
Reagent Composition
|
Components |
24T |
96T |
|
Frag/Elute Buffer |
168 μL |
672 μL |
|
RT Strand Specificity Reagent |
96 μL |
384 μL |
|
First Strand Synthesis Enzyme Mix |
48 μL |
192 μL |
|
Second Strand & dA Buffer |
240 μL |
960 μL |
|
Second Strand & dA Buffer with dUTP |
240 μL |
960 μL |
|
Second Strand & dA Enzymes |
120 μL |
480 μL |
|
Nuclease-free Water |
2 mL |
8 mL |
|
Ligation Buffer |
396 μL |
1584 μL |
|
Ligase Mix T5 |
72 μL |
288 μL |
|
2X PCR Master Mix |
600 μL |
1.2mL X 2 |
|
10X ILM PCR Primers |
120 μL |
480 μL |
|
MGI PCR Primer Mix |
120 μL |
480 μL |
|
Low EDTA TE |
1 mL X 2 |
10 mL |
Note 1. The kit provides both Second Strand & dA Buffer and Second Strand & dA Buffer with dUTP. When selecting non-strand-specific library construction, please use
Second Strand & dA Buffer; when selecting strand-specific library construction, please use Second Strand & dA Buffer with dUTP.
Note 2. This kit contains MGI PCR Primer Mix (for MGI platform) and 10X ILM PCR Primers (for Illumina platform).
Storage Conditions
Store at -25~-15℃.
Notes
1. RNA sample quality control
1.1 This kit is suitable for total RNA starting amount of 10-1000 ng. It is recommended to use Qubit RNA HS Assay Kit (Thermo Fisher SCIENTIFIC, Cat. Q32855) to quantify total RNA. Too low input amount will affect normal library construction.
1.2 When using poly (A) mRNA capture reagent for RNA enrichment, it must be a high-quality total RNA sample (RIN value>7), otherwise the library construction will introduce 3' end preference. Therefore, for RNA samples with RIN value <7, it is recommended to use rRNA Depletion Module series products for RNA enrichment.
1.3 For plant or other eukaryotic cell RNA samples, if the RNA is degraded but 28S and 18S bands can be seen in agarose gel, it is recommended to try to increase the total RNA input amount and appropriately increase the number of PCR cycles to obtain a sufficient amount of library.DNA Fragmentation.
2. Principles of using magnetic beads
2.1 Take out the magnetic beads half an hour before use and place them at room temperature for equilibrium.
2.2 Avoid storing 2X Oligo (dT)25 Capture Beads and AFTMag NGS DNA Clean Beads at -20℃. Freezing will cause the magnetic beads to aggregate and cannot be dispersed again, resulting in the failure of the magnetic beads. If the magnetic beads are frozen, it is recommended to purchase them separately.
2.3 During the purification process of AFTMag NGS DNA Clean Beads, Low EDTA TE must be added after the alcohol is completely evaporated, that is, the color of the magnetic beads changes from bright brown to frosted brown. If the alcohol is not completely evaporated or the magnetic beads are too dry (become cracked), the library yield will be affected.
3. Library quality control
3.1 If the library peak is smooth and has no abnormal burrs, and there is no detection peak at 130bp (adapter dimer), and there is no large-scale fragment peak on the right side of the library peak, it can be preliminarily judged that the library construction is successful.
3.2 The quality of Total RNA is qualified (RIN value>7), and the library construction is normal. The possible reasons and solutions for unsuccessful library construction are as follows:The RIN value of Total RNA is a quality assessment of Total RNA, and it cannot fully represent the abundance and integrity of poly (A) RNA. For some special samples, although the integrity of Total RNA is good, many mRNAs are degraded, resulting in a large loss of poly (A) RNA during purification, and thus the library construction fails. If you encounter this situation, you can purify the poly (A) RNA and evaluate its abundance and integrity. The method is to add 6 μL of Tris Buffer after the end of step 7.1.10 of the experimental process, heat it at 80℃ for 2 minutes, place it on a magnetic rack, and take the supernatant after clarification, which is the complete poly (A) RNA. Then take 1 μL for Agilent 2100 RNA 6000 Pico chip analysis.
3.3 According to the analysis results, if the mRNA abundance is low, the amount of Total RNA input for library construction can be increased. If the mRNA integrity is poor, the mRNA interruption time can be adjusted.
4. About library construction adapters
4.1 HYASEN can provide long adapters and short adapters, customers can choose according to experimental requirements.
4.2 If you construct a PCR-Free library, you need to use long adapters with complete indexes in the adapter ligation step, and remove excess adapters from the adapter ligation product before sequencing.
4.3 This kit is compatible with Full Adapter and Truncated Adapter of MGI and Illumina sequencing platforms.
4.4 The quality and concentration of adapters directly affect the ligation efficiency and library yield. Too high an adapter dosage may produce more adapter dimers; too low an adapter dosage may affect the ligation efficiency and library yield. Table 1 lists the recommended adapter concentrations for this reagent and different Total RNA starting amounts.
Table 1. Recommended concentrations for adapter
|
Total RNA |
Illumina adapter |
MGI adapter |
|
1μg |
15 μM |
15 μM |
|
100 ng |
3 μM |
7.5 μM |
|
10 ng |
1.5 μM |
3 μM |