Product Introduction
This kit is suitable for the construction of new RNA transcriptome libraries on the Illumina platform. It is simple to operate, has a short library construction time, and has stable results. It is suitable for the construction of new RNA libraries for various samples such as living animals, plants, and living cells. This kit uses 5-ethynyl uracil (EU) to label new RNA in living cells, and then uses click chemistry to label the new RNA with biotin, and then uses streptavidin magnetic beads to specifically capture the new RNA, and finally constructs a library for the new RNA. Combined with the experimental time design, it can quantitatively analyze the newly generated RNA, and at the same time, it can study the RNA transcription level and degradation issues.
Features
Kit 1: Store at 2~8℃, valid for 12 months;
Kit 2: Store at -25~-15℃, valid for 12 months;
Kit 3: Store at -25~-15℃, valid for 12 months.
1.1 EU incubation of cell samples
When the cell density is 70-90% or the cell density meets the requirements of the experimental design, EU incubation is performed. Use complete culture medium to dilute EU. The final concentration of common cell EU is 1 mM and incubate for 2 hours. Or according to literature reports, the final concentration of EU and incubation time can also be adjusted. For the first experiment, a preliminary experiment can be performed according to the recommended EU dose and time; for example, after the cells in the T25 flask have their culture medium removed, the prepared culture medium containing 1 mM EU is added to the culture flask and incubated for 2 hours. * Follow step 2 for subsequent experiments. The following table shows the recommended EU dose and time for testing:
1.2 Animal tissue EU incubation
According to the animal's body weight, the injection or gavage can be performed. EU can be prepared with PBS to a certain concentration at a dosage of 200-400 mg/kg, and then injected or gavaged. The specific dosage is related to the type of animal used, weight and method of use. You can refer to relevant literature. For example, it is recommended to explore the concentration of EU for the first time, or directly use a concentration of 200 mg/kg for testing. After 4 hours, * Or after an appropriate time determined by the specific experiment, quickly kill the mouse and remove the desired tissue. Follow step 2 in 1 for subsequent experiments.
*Experiments have been verified on common animal models such as mice, rats, ground squirrels, and rabbits.
1.3 EU incubation of plant samples
Add EU for incubation according to the culture environment of the plant. The dosage can be determined by referring to relevant literature. For liquid culture, EU can be added to the culture medium to a final concentration of 0.5 mM and cultured for 6-8 hours. * You can extract RNA according to your own method. After extracting RNA, proceed to the second step of the click reaction follow-up experiment.
*Experiments have verified the hydroponic and soil-cultured ginseng, various flowers and food crops, etc.
2.1 For cultured cell samples, remove the culture medium, lyse the cells with 1 ml TRIZOL, place in an enzyme-free EP tube, and let stand at room temperature for 5 min; for plant and animal samples, after obtaining the corresponding tissue, wash the tissue 2-3 times with enzyme-free PBS, place it in 1 mL TRIZOL and grind it with a grinding instrument until no tissue blocks are visible, place it in an enzyme-free EP tube, and let stand at room temperature for 5 min;
2.2 Add 200 μL chloroform, vortex to mix, and let stand at room temperature for 3 min;
2.3 Centrifuge at 14000g for 5 min, take the supernatant, add an equal volume of isopropanol, and vortex to mix;
2.4 Place on ice for 10 min, centrifuge at 14000g for 20 min, and discard the supernatant;
2.5 Wash the precipitate with 75% ethanol and centrifuge at 14000g for 5 min;
2.6 Repeat step 6;
2.7 Discard the supernatant, wait for the ethanol to evaporate, and add 50 μL of DEPC water to dissolve the RNA.
2.8 rRNA removal: It is recommended to use the Qihengxing Ribosomal RNA Removal Kit to remove rRNA (this step is a recommended option, rRNA removal can improve the detection sensitivity of the target RNA and reduce the amount of sequencing).
3.1 Click reaction solution preparation
3.2 Add 150 μL of click reaction solution to each sample and vortex to mix;
3.3 Place on ice for 30 min;
3.4 Add 1 ml TRIZOL to the click reaction solution and vortex to mix;
3.5 Repeat the RNA extraction process (TRIZOL extraction).
4.1 Vortex to fully suspend the streptavidin magnetic beads. Do not centrifuge to prevent the magnetic beads from settling.
4.2 Take 20 μL of resuspended magnetic beads from each sample and place them in a new EP tube. For the cleaning of SMB magnetic beads required for multiple samples, they can be done together, such as dispensing the magnetic beads of 6 samples (120 μL) or 12 samples (240 μL) into one tube for cleaning. Add 20 μL of 2×Wash Buffer to each 20 μL of streptavidin magnetic beads, mix well, place on a horizontal rotator and rotate at low speed for 1 min, then place on a magnetic stand and wait for the magnetic beads to be adsorbed, and discard the supernatant;
4.3 After removing the EP tube containing magnetic beads, add 40 μL of 2×Wash Buffer to every 20 μL of streptavidin magnetic beads, mix well, and place on a horizontal rotator for low speed rotation for 1 min. After placing on a magnetic stand and completing the magnetic beads adsorption, discard the supernatant;
4.4 Perform the same operation as step 4.2, washing each 20 μL of streptavidin beads twice with 40 μL of solution A, and then washing twice with 40 μL of solution B;
4.5 After discarding the supernatant, add 50 μL of 2× Wash Buffer to the magnetic beads (each sample) to resuspend;
5.1 Towards 3 RNA obtained in step 1 was added to each sample with 50 μL of the prepared streptavidin magnetic beads;
5.2 After vortex mixing, incubate at room temperature for 20 min, place the EP tube in the magnetic rack for 2-3 min, and discard the supernatant after the magnetic beads are adsorbed;
5.3 Add 20 μL of 1× Wash Buffer to each sample, mix well, and rotate on a horizontal rotator at low speed for 1 min. Place on a magnetic rack and wait for the magnetic beads to be adsorbed. Discard the supernatant. Repeat twice.
6.1 Place the sample back into the magnetic rack, let it stand at room temperature for 5 min, and remove all the supernatant.
【Note】: Finally, use a 10 μL pipette to suck out the remaining liquid;
6.2 Remove the sample from the magnetic stand, resuspend the magnetic beads with 18 μL Frag/Prime Buffer, and pipette up and down 6 times to mix thoroughly; place the sample in a PCR instrument (preset to 94°C) and incubate at 94°C for 7 min.
6.3 Enter the first chain synthesis.
7.1 Take out the first-strand synthesis reagent from -20°C, invert and mix, and centrifuge. Prepare the first-strand cDNA synthesis reaction solution as shown in the table below.
7.2 Use a pipette to gently pipette or vortex to mix, and then centrifuge the reaction solution to the bottom of the tube.
7.3 Place the above PCR tube in a PCR instrument, set the reaction program as shown in the table below, and synthesize the first-strand cDNA. After the reaction is completed, immediately synthesize the second-strand cDNA.
8.1 Take out the second-strand synthesis reagent from -20 °C, thaw it and mix it by inversion; prepare the second-strand cDNA synthesis/end repair/addition A reaction solution as shown in the table below.
8.2 Use a pipette to gently pipette to mix, and then centrifuge the reaction solution to the bottom of the tube.
8.3 Place the above PCR tube in a PCR instrument and set the reaction program as shown in the table below to synthesize the second-strand cDNA.
This step allows the specific Illumina® adapters to be connected to the ends of the end-repaired and dA-tailed products. Thaw each reagent in the table below, mix by inversion, and place on ice for later use.
9.1 After completing the above steps, continue to prepare the reaction system shown in the table below in the PCR tube.
9.2 Use a pipette to gently pipette to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.
9.3 Place the PCR tube in the PCR instrument and set the reaction program as shown in the table below to perform the adapter ligation reaction:
10.1 Vortex or invert the beads thoroughly to ensure mixing.
10.2 Add 20 μL (0.20 ×) of the first round of sorting magnetic beads to the above 100 μL ligation system, vortex or pipette 10 times to mix, and incubate at room temperature for 10 min.
10.3 Centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clear (about 5 min), carefully transfer the supernatant to a clean centrifuge tube.
10.4 Add 0.10× (120 μL × 0.1=12 μL) of the second round of sorting magnetic beads to the supernatant.
10.5 Vortex or pipette up and down 10 times to mix, and let stand at room temperature for 10 min.
10.6 Centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clear (about 3 min), carefully remove the supernatant.
10.7 Keep the PCR tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant.
10.8 Repeat step 7.
10.9 Keep the PCR tube in the magnetic rack at all times, open the lid and dry the magnetic beads until cracks just appear (about 3 minutes).
10.10 Take the PCR tube out of the magnetic stand and add 21 μL ddH 2 O, vortex or use a pipette to mix thoroughly, and let it stand at room temperature for 5 min.
10.11 Centrifuge the PCR tube briefly and place it on a magnetic stand to separate the beads and liquid. After the solution is clear (about 3 min), carefully transfer 20 μL of the supernatant to a clean tube.
This step will enrich the adapter ligation products after purification or length sorting by PCR amplification.
11.1 Thaw the reagents in the table below, mix thoroughly by inversion, and place on ice for later use.
11.2 Prepare the reaction system shown in the following table in a sterile PCR tube.
11.3 Use a pipette to gently pipette or shake to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube.
11.4 Place the PCR tube in the PCR instrument, set the indicated reaction program, and perform PCR amplification.
12.1 Preparation: Take StarPure DNA Size Selection Kit-V2 out of the refrigerator and equilibrate it at room temperature for at least 30 min. Meanwhile, prepare 80% ethanol.
12.2 Vortex or invert the beads thoroughly to ensure thorough mixing.
12.3 Pipette 45 μL of Beads (0.9×, Beads: DNA=0.9:1) into the Adapter Ligation product, vortex or pipette to mix, and incubate at room temperature for 5 min.
12.4 Briefly centrifuge the EP tube and place it on a magnetic stand to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant.
12.5 Keep the EP tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.
12.6 Repeat step 5 for a total of two rinses.
12.7 Keep the EP tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until cracks just appear (no more than 5 minutes).
12.8 Take the EP tube out of the magnetic stand and add 21 μL ddH 2 O, vortex or use a pipette to gently blow until fully mixed, and let it stand at room temperature for 5 minutes. Centrifuge the EP tube briefly and place it on a magnetic stand until the solution is clear (about 3 minutes);
12.9 Carefully transfer 20 μL of supernatant to a new EP tube for library quantification and quality control.
