The scmRNA Amplication Kit simplifies and accelerates complete transcriptome amplification of total RNA or mRNA from single cells. This kit can create 2ng-20ng cDNA amplification products from 1-500 cells or 10 pg-10 ng total RNA as a template for library creation using 2.5 l sample volumes, making it suitable for transcript discovery and differential expression. The kit comes with all of the necessary enzymes and buffers for cell lysis, reverse transcription, and cDNA amplification. Oligo(dT) VN is used as a reverse transcription primer for the first-strand cDNA synthesis in the Kit. The 3'-end of the first-strand cDNA will be cleaved to a special adapter during reverse transcription. This one-of-a-kind adapter sequence can be used to amplify full-length cDNA without rRNA contamination or 3' bias. All Kit parts are handled to thorough quality check to guarantee a reliable and reproducible library preparation.
Storage:
Box 1 should be stored at -70°C. Box 2 should be stored at -20°C.
Components:
Specifications:
| Sample amount | 1-500 cells or 10 pg -10 ng of purified total RNA |
| Features | A single cell or 10 pg total RNA can be used as template in this procedure, with high amplification efficiency. Optimized reaction system for high amplification sensitivity increases the detection efficiency of low expression genes. Full-length cDNA will be amplified by double-ended primers avoiding the 3’and 5’ bias and rRNA contamination. |
| Application | It is recommended to use this kit for: Regulatory RNA sequencing Gene expression profiling Transcript detection |
| Species Category | mammalian, other eukaryotic |
| Sample type | cells without cell wall or purified total RNA with poly A sequence; not suitable for prokaryotic cells or fixed cells. |
| Sequencing Platform | Illumina |
First Strand cDNA Synthesis (Please operate in an Ultra-Clean workbench)
The 1st strand cDNA was synthesis from purified RNA or total RNA of cells as template at this step. Take Oligo(dT) VN as reverse transcription primer, and use the Template-switching activity of Sc Reverse Transcriptase to ligate an adapter to the 3'-end of full-length cDNA.
- Take out all of the components for 1st strand cDNA synthesis, dissolve them on ice, mix by vortex and briefly centrifuge to collect them to the bottom of the tube, and then place on ice.
- Sample Buffer preparation.
- Sample Preparation.
- Prepare an annealing reaction solution.
- Mix thoroughly by gently pipetting, briefly centrifuge, and place on ice.
- Preheat the PCR instrument (with hot-lid) to 72°C, and run the program.
- Prepare the reverse transcription reaction solution.
- Mix thoroughly by gently pipetting, briefly centrifuge, and place on ice.
- Place the PCR tube to the preheated (42°C) PCR instrument, and run the program.
Full-length cDNA Amplification (Please operate in an Ultra-Clean workbench)
The 1st Strand cDNA synthesized during the previous step is amplified through PCR in this step. Take out the components for PCR, dissolve them on ice, mix thoroughly by vortex and briefly centrifuge to collect them to the bottom of the tube, and then place on ice.
- Prepare the PCR amplification reaction solution.
- Mix thoroughly by gently pipetting, briefly centrifuge, and place on ice.
- Run PCR program on a thermocycler.
- Place the products on ice after reaction.
Purification and Detection of cDNA Amplification Products
The cDNA amplification products are purified by beads in this step, and the qualities are detected by Aglient 2100 Bioanalyzer.
Aliquot CD DNA Clean Beads into 1.5 ml EP tubes accordingly. Mix the CD DNA Clean Beads by vortex and incubate at room temperature for 30 min. Prepare fresh 80% ethyl alcohol (approximately 400 µl for each sample).
- Mix CD DNA Clean Beads thoroughly by vortex, add 25 µl of CD DNA Clean Beads to the cDNA amplification products, pipette 10 times to mix the reaction system thoroughly.
- Incubate at room temperature for 8 min to let the cDNA bind on the beads.
- Briefly centrifuge the tube and place it on a magnetic stand to separate the beads from the solution. Keep the PCR tube on the magnetic stand. Wait until the solution clarifies (about 5 min). Keep on magnetic stand, and carefully discard the supernatant without disturbing the beads.
- Keep the PCR tube on the magnetic stand, add 200 µl of freshly prepared 80% ethanol to rinse the beads. DO NOT re-suspend the beads! Incubate for 30 sec at room temperature and carefully discard the supernatant without disturbing the beads.
- Repeat Step 4 (rinse the beads twice in total).
- Keep the samples on the magnetic stand, open the tube and air-dry the beads for 5-10 min.
- Take the sample out of the magnetic stand. Add 17 µl of Elution Buffer to cover the beads, mix them by pipetting. Incubate at room temperature for 2 min. If the beads are over-dried and cracking, please extend the incubation time.
- Briefly centrifuge the PCR tube and then place on the magnetic stand to separate the beads and solution until the solution are clarified (about 5 min).
- Carefully transfer 15 µl of supernatant to a new low adsorption EP tube. Store at -20°C.
- Detection of cDNA Amplification Products
Library Preparation for Sequencing on Illumina® Platforms
At this step, it is recommended to take 1 ng of cDNA as starting material, and using CD NEXT DNA Library Prep Kit for Illumina (DP002) or equivalent for library construction.
