Ribosomal RNA Depletion for Massively Parallel RNA Sequencing Protocol

Preparing Total RNA

Perform Dnase treatment of the total RNA to remove DNA contamination. Total RNA is isolated using RNA Kit or TRIzol® Reagent. Resuspend isolated total RNA in DEPC-treated water accordingly. Check the quality of total RNA.

Hybridization Step

1. Set a water bath or heat block to 70-75°C.
2. To a sterile, RNase-free 1.5-mL microcentrifuge tube, add Total RNA (0.5-10 mg) in less than 10 mL, 8 mL bacterial 16S and 23S probe sets (12.5 pmol/mL), and 100 mL Hybridization Buffer.
3. Incubate the tube at 70-75°C for 5 min to denature RNA.
4. Allow the sample to cool to 37°C slowly over a period of 30 min by placing the tube in a 37°C water bath. Do not cool samples quickly by placing tubes in cold water.

Preparing Beads

1. Resuspend Magnetic Beads in its original bottle by thorough vortexing.
2. Pipette 750 mL of the bead suspension into a sterile, RNase free, 1.5 mL microcentrifuge tube.
3. Place the tube with the bead suspension on a magnetic separator for 1 min. The beads will settle to the tube side that faces the magnet. Gently aspirate and discard the supernatant.
4. Add 750 mL DEPC water to the beads and resuspend the beads by slow vortexing.
5. Place the tube on a magnetic separator for 1 min. Aspirate and discard the supernatant.
6. Repeat steps 4 and 5 once.
7. Resuspend the beads in 750 mL Hybridization Buffer and transfer 250 mL beads to a new tube and maintain the tube at 37°C for use at a later step.
8. Place the tube with 500 mL beads on a magnetic separator for 1 min. Aspirate and discard the supernatant.
9. Resuspend beads in 200 mL Hybridization Buffer and keep the beads at 37°C until use.

Removing rRNA

1. After the incubation at 37°C for 30 min of the hybridized sample (above), briefly centrifuge the tube to collect the sample to the bottom of the tube.
2. Transfer the sample (approximately 118 mL) to the prepared Magnetic beads. Pipette up and down or low-speed vortex to mix well.
3. Incubate the tube at 37°C for 15 min. During incubation, gently mix the contents occasionally. Centrifuge the tube to collect the sample.
4. Place the tube on a magnetic separator for 1 min to pellet the rRNA complex. Do not discard the supernatant. The supernatant contains RNA.
5. Place the tube with 250 mL beads from step 7 on a magnetic separator for 1 min. Aspirate and discard the supernatant.
6. To this tube of beads, add approximately 318 mL of supernatant containing RNA. Pipette up and down or low-speed vortex to mix well.
7. Incubate the tube at 37°C for 15 min. During incubation, gently mix the contents occasionally. Collect the sample to the bottom of the tube by briefly centrifuging.
8. Place the tube on a magnetic separator for 1 min to pellet the rRNA-probe complex. Do not discard the supernatant as the supernatant contains RNA.
9. Transfer the supernatant containing RNA to a new tube.

Concentrating

Concentrating RNA Using Ethanol Precipitation

1. Transfer the RNA sample into a clean, RNase free 1.5 mL or 2 mL microcentrifuge tube.
2. Add 1 mL Glycogen (20 mg/mL), 1/10 Sample (eluted RNA) volume of 3 M sodium acetate, 2.5× Sample volumes of 100% ethanol to the following components to RNA.
3. Mix well and incubate at −80°C for at least 30 min.
4. Centrifuge the tube for 15 min at 12,000 × g at 4°C. Carefully discard the supernatant without disturbing the pellet.
5. Add 500 mL 70% cold ethanol.
6. Centrifuge the tube for 5 min at 12,000 × g at 4°C. Discard the supernatant without disturbing the pellet.
7. Repeat steps 5 and 6 once.
8. Air-dry the pellet for approximately 5 min. Resuspend the RNA pellet in 10-30 mL DEPC-treated water.
9. Place RNA on ice or store RNA at -80°C until use.

Reference:

1. Chen Z, Duan X. Ribosomal RNA depletion for massively parallel bacterial RNA-sequencing applications[M]//High-Throughput Next Generation Sequencing. Humana Press, Totowa, NJ, 2011: 93-103.