The Illumina mRNA-seq Library Prep Kit makes library preparation for next-generation sequencing (NGS) applications on Illumina platforms quick and easy. This kit allows library preparation from as little as 10ng to 4ug total RNA from animals, plants, fungi, and other eukaryotes with high purity and integrity. This kit includes three packages: NR1, NR21, and NR31, each containing the enzymes and buffers needed for mRNA enrichment, fragmentation, and cDNA synthesis, as well as end-repair, adapter ligation, and amplification. Magnetic beads can be used to quickly select sizes, allowing you to meet the needs of different experiments. All kit components are subjected to rigorous quality control to ensure that library preparation is consistent and repeatable.
Storage:
NR 1 should be stored at 2-8°C. NR 21 should be stored at -20°C. NR 31 should be stored at -20°C
Components:
Specifications:
| Sample amount | 10ng-4µg |
| Features | Fast and convenient workflow. Enables higher library conversion rate, higher and uniform coverage yields. |
| Application | It is recommended to use this kit for: Gene expression analysis Single nucleotide variation calling Gene fusion detection Transcript Variants Novel Transcripts Target transcriptome analysis |
| Species Category | animals, plants, fungus, and other eukaryotes |
| Sample type | total RNA with good purity and integrity |
| Sequencing Platform | Illumina |
Workflow
Workflow of CD mRNA-seq Library Prep Kit for Illumina ®
Protocol
mRNA Purification and Fragmentation
- Take NR1 (mRNA Capture Beads, Beads Wash Buffer, Tris Buffer, and Beads Binding Buffer) from 2 - 8℃, set sit and equilibrate to room temperature.
- Prepare the RNA sample carefully: dissolve 0.01 μg – 4 μg of total RNA in 50 μl Nuclease-free H2O in a Nuclease-free PCR tube. Keep the tube on ice and proceed to the next step as soon as possible.
- Softly suspend RNA Capture Beads thoroughly by inverting, aspirate 50 μl beads into prepared RNA sample, mix thoroughly by pipetting up and down for 10 times.
- Two rounds of mRNA Purification to guarantee the removal efficacy of rRNA.
- Take the sample out of the magnetic stand, add 19.5 μl of Frag/Prime Buffer to resuspend the beads thoroughly by pipetting up and down for 10 times. Incubate the sample in a PCR device and set programs according to the fragment size required.
- Place the sample on the magnetic stand. Wait until the solution clarifies (about 5 min), and carefully aspirate 17 μl of supernatant into a new Nuclease-free PCR tube, then immediately proceed to synthesis of first-strand cDNA.
Synthesis of Second Strand cDNA
- Thaw the 1st Strand Buffer (from -20℃) and mix thoroughly by inverting the tube. Prepare the reaction solution to synthesize the first strand cDNA.
- Mix thoroughly by gently pipetting up and down for 10 times.
- Put the sample in a PCR instrument and run program (first strand cDNA synthesis).
- Thaw the 2nd Strand Buffer and mix thoroughly by inverting. Prepare the reaction solution to synthesize the 2nd strand cDNA.
- Mix thoroughly by gently pipetting up and down for 10 times.
- Put the sample in a PCR instrument and run program (2nd strand cDNA synthesize).
- Purification of the double strand cDNA.
End Repair
- Thaw the End Prep Mix 3 and mix thoroughly by inverting the tube. Prepare the reaction solution.
- Mix thoroughly by gently pipetting up and down for 10 times.
- Put the sample in a PCR instrument and run program for end-repair.
Adapter Ligation
- Thaw the RNA Adapter and mix thoroughly by inverting the tube.
- Thaw the Rapid Ligation Buffer and mix thoroughly by inverting the tube. Keep on ice and proceed to the next step.
- Prepare the reaction solution.
- Mix thoroughly by gently pipetting up and down for 10 times.
- Put the sample in a PCR instrument and run program for adapter ligation.
Purification and Size Selection of Adapter-ligated DNA
This step provides two options. Please select carefully according to your needs:
Option A: Two-round purification were performed without sorting operation. This option can effectively obtain inserts of 150 bp - 200 bp at 94°C for 8 min, and remove the residual adapters.
Option B: One round of purification and then followed by two-round sorting; 200 bp of inserts can be obtained according to the different sorting conditions in Table 2 without residual adapters.
Library Amplification
- Thaw the PCR Primer Mix and Amplification Mix 1 thoroughly by inverting the tube. Prepare the reaction solution.
- Put the sample in a PCR instrument and run PCR program.
- Purification of the PCR product with CD DNA Clean Beads.
- Library Quality Determination.
