Product Introduction
This kit is suitable for constructing nascent RNA transcriptome libraries for the Illumina platform. It features simple operation, short library construction time, and stable results. It is suitable for constructing nascent RNA libraries from a variety of samples, including living animals, plants, and living cells. This kit uses 5-ethynyluracil (EU) to label nascent RNA in living cells. The nascent RNA is then labeled with biotin through a click chemistry reaction. The nascent RNA is then specifically captured using streptavidin magnetic beads, and the library is constructed. Combined with the experimental timeline, this kit enables quantitative analysis of newly generated RNA and allows for the study of RNA transcription levels and degradation.
Features
Kit 1: Store at 2-8°C, 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 EU used in common cells is 1 mM and incubate for 2 hours. Alternatively, the final concentration of EU and incubation time can be adjusted according to literature reports. For the first experiment, a preliminary experiment can be performed according to the recommended EU dose and time; for example, after removing the culture medium of the cells in the T25 flask, add the prepared culture medium containing 1 mM EU to the culture flask and incubate for 2 hours. * Follow step 2 for subsequent experiments. The following table shows the recommended EU dose and time for testing:
1.2 EU incubation of animal tissues
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 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, when using for the first time, it is recommended to explore the concentration of EU or directly use a concentration of 200 mg/kg for testing. After 4 hours, * Alternatively, after an appropriate time determined for the specific experiment, quickly sacrifice the mouse and remove the desired tissue. Proceed with subsequent experiments according to step 2 in step 1.
*Experimentally verified in 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.
*Experimentally verified for hydroponic and soil-cultured ginseng, various flowers and grain crops, etc.
2.1 For cultured cell samples, remove the culture medium and lyse the cells with 1 ml of TRIZOL. Place the cells in an enzyme-free EP tube and let them stand at room temperature for 5 minutes. For plant and animal samples, obtain the corresponding tissue, wash the tissue 2-3 times with enzyme-free PBS, place the tissue in 1 ml of TRIZOL and grind it with a grinding instrument until no tissue fragments are visible. Place the tissue in an enzyme-free EP tube and let it stand at room temperature for 5 minutes.
2.2 Add 200 μL of chloroform, vortex to mix, and let stand at room temperature for 3 min;
2.3 Centrifuge at 14000g for 5 min, remove the supernatant, add an equal volume of isopropanol, and vortex to mix;
2.4 Place on ice for 10 min, centrifuge at 14,000 g for 20 min, and discard the supernatant.
2.5 Wash the pellet with 75% ethanol and centrifuge at 14,000 g for 5 min.
2.6 Repeat step 6;
2.7 Discard the supernatant. After the ethanol evaporates, add 50 μL of DEPC water to dissolve the RNA.
2.8 rRNA removal: The Qihengxing Ribosomal RNA Removal Kit is recommended for rRNA removal (this step is recommended. rRNA removal can improve the detection sensitivity of target RNA and reduce the sequencing amount).
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 of TRIZOL to the click reaction solution and vortex to mix.
3.5 Repeat the RNA extraction process (TRIZOL extraction).
4.1 Vortex the solution to fully suspend the streptavidin magnetic beads. Do not centrifuge to prevent the beads from settling.
4.2 Transfer 20 μL of resuspended magnetic beads to a new EP tube for each sample. For SMB magnetic beads required for multiple samples, wash them together, for example, by aliquoting the magnetic beads from 6 samples (120 μL) or 12 samples (240 μL) into one tube for washing. Add 20 μL of 2× Wash Buffer to each 20 μL of streptavidin magnetic beads, mix thoroughly, and rotate on a horizontal rotator at low speed for 1 minute. Place on a magnetic stand until the beads are adsorbed, and discard the supernatant.
4.3 After removing the EP tube containing the magnetic beads, add 40 μL of 2× Wash Buffer for every 20 μL of streptavidin magnetic beads, mix thoroughly, and rotate on a horizontal rotator at low speed for 1 min. After placing on a magnetic stand and waiting for the magnetic beads to adsorb completely, discard the supernatant.
4.4 Repeat step 4.2, but wash each 20 μL of streptavidin beads twice with 40 μL of solution A, and then wash 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) and resuspend;
5.1 Towards 3 For the RNA obtained in step 1, add 50 μL of prepared streptavidin magnetic beads to each sample;
5.2 After vortex mixing, incubate at room temperature for 20 minutes. Place the EP tube in a magnetic rack for 2-3 minutes. After the magnetic beads are adsorbed, discard the supernatant.
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 magnetic beads to adsorb. Discard the supernatant and repeat twice.
6.1 Place the sample back into the magnetic rack, let it stand at room temperature for 5 minutes, and remove all the supernatant.
【Note】: Finally, use a 10 μL pipette to aspirate the remaining liquid;
6.2 Remove the sample from the magnetic stand, resuspend the magnetic beads in 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 first-strand synthesis.
7.1 Remove the first-strand synthesis reagent from -20°C, invert and mix thoroughly, and then centrifuge briefly. 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 tubes in a PCR instrument and set the reaction program according to the table below to synthesize the first-strand cDNA. Immediately after the reaction is completed, proceed with the synthesis of the second-strand cDNA.
8.1 Remove the second-strand synthesis reagent from -20°C, thaw, and mix thoroughly 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 according to the table below to synthesize the second-strand cDNA.
This step allows for ligation of specific Illumina® adapters to the ends of the end-repaired and dA-tailed products. Thaw the reagents in the table below, mix thoroughly by inversion, and place on ice until ready to use.
9.1 Prepare the reaction system shown in the table below in the PCR tube after completing the above steps.
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 of the first-round sorting magnetic beads (0.20 ×) to the 100 μL ligation system above. Vortex or pipette 10 times to mix thoroughly. Incubate at room temperature for 10 minutes.
10.3 Briefly centrifuge the PCR tube and place it on a magnetic rack. After the solution has clarified (approximately 5 minutes), carefully transfer the supernatant to a clean centrifuge tube.
10.4 Add 0.10× (120 μL × 0.1=12 μL) of second-round separation magnetic beads to the supernatant.
10.5 Vortex or pipette up and down 10 times to mix thoroughly, and let it stand at room temperature for 10 minutes.
10.6 Briefly centrifuge the PCR tube and place it on a magnetic rack. After the solution has clarified (approximately 3 minutes), carefully remove the supernatant.
10.7 Keeping 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 seconds 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 they just begin to crack (approximately 3 minutes).
10.10 Remove the PCR tube from the magnetic stand and add 21 μL ddH 2 Vortex or pipette gently to mix thoroughly, and let it stand at room temperature for 5 min.
10.11 Briefly centrifuge the PCR tube and place it on a magnetic stand to separate the beads and liquid. After the solution has clarified (approximately 3 minutes), carefully transfer 20 μL of the supernatant to a clean tube.
This step involves PCR amplification and enrichment of the purified or length-sorted adapter-ligated products.
11.1 Thaw the reagents in the table below, mix thoroughly by inversion, and place on ice until ready for use.
11.2 Prepare the reaction system shown in the table below in a sterile PCR tube.
11.3 Use a pipette to gently pipette or oscillate 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 reaction program as shown below, and perform PCR amplification.
12.1 Preparation: Take the StarPure DNA Size Selection Kit-V2 out of the refrigerator and equilibrate it at room temperature for at least 30 minutes. At the same time, 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 (approximately 5 minutes), carefully remove the supernatant.
12.5 Keep the EP tube in the magnetic rack and 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 Remove the EP tube from the magnetic stand and add 21 μL ddH 2 Vortex or pipette gently to mix thoroughly, then let stand at room temperature for 5 minutes. Briefly centrifuge the EP tube and place it on a magnetic stand until the solution clears (approximately 3 minutes).
12.9 Carefully transfer 20 μL of supernatant to a new EP tube for library quantification and quality control.
