NGS RNA Fragmentation Module

The NGS RNA Fragmentation Module is optimized to fragment RNA into small pieces using divalent cations under elevated temperature.

Applications

• RNA fragmentation.

RNA Fragmentation Protocol

This protocol is used to fragment 10-400 ng of rRNA-depleted, or poly(A)-enriched RNA.

1. Assemble the following reaction in a microcentrifuge tube:

     Purified RNA                                                   10-400 ng   
     RNA Fragmentation Buffer (10×)                    2 µL
     Nuclease-free water                                        to 20 µL
    Total volume                                                    20 µL

2. Place the tubes in a thermocycler and carry out the fragmentation as follows:

3. Place the tubes on ice and proceed immediately to 1st strand synthesis. Or add 2 µL of RNA Fragmentation Stop Solution (10×), and proceed RNA clean up.

Clean Up Fragmented RNA Using Ethanol Precipitation

1. Mix the following components in a microcentrifuge tube:

     Fragmented RNA from Step 3                          22 µL
     3 M Sodium Acetate, pH 5.2                            2 µL
     Linear Acryamide, 10 mg/mL                           1 µL
     100% Ethanol                                                   60 µL
     Total volume                                                     85 µL

2. Incubate at -80°C for 30 minutes.

3. Centrifuge at 14,000 rpm for 25 minutes at 4°C in a microcentrifuge.

4. Carefully remove ethanol.

5. Wash pellet with 300 μL of 70% ethanol.

6. Centrifuge and carefully remove 70% ethanol.

7. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.

8. Resuspend in 20 μL Nuclease-free Water.