Deplete rRNA

•

DB1 (Depletion Probe Buffer) (blue cap)

•

DP1 (Depletion Probe Pool) (blue cap)

•

DPM (Microbiome Depletion Probe Pool) (clear cap) Only the Ribo-Zero Plus Microbiome kit (cat# 20072063) contains DPM.

•

ELB (Elution Buffer)

•

PRB (Probe Removal Buffer) (red cap)

•

PRE (Probe Removal Enzyme) (red cap)

•

RDB (RNA Depletion Buffer) (yellow cap)

•

RDE (RNA Depletion Enzyme) (yellow cap)

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Agencourt RNAClean XP

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Freshly prepared 80% ethanol (EtOH)

•

Nuclease-free ultrapure water

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1.7 ml microcentrifuge tubes, RNase-free (3)

•

96-well PCR plates, semiskirted (2)

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Microseal 'B' adhesive film

•

Prepare for a later procedure:

–

EPH3 (Elute, Prime, Fragment 3HC Mix)

Prepare 80% EtOH fresh and discard after one day. The protocol has four cleanup steps that require fresh 80% EtOH.

1.

Prepare the following consumables:

2.

Prepare 80% EtOH from absolute EtOH.

3.

Save the following HYB_DP1 program on the thermal cycler:

•

Choose the preheat lid option and set to 100°C

•

Reaction volume is 15 µl

•

95°C for 2 minutes

•

Decrease 0.1°C per second until the temperature reaches 37°C

•

Hold at 37°C

4.

Save the following RNA_DEP program on the thermal cycler:

•

Choose the preheat lid option and set to 100°C

•

Reaction volume is 20 µl

•

37°C for 15 minutes

•

Hold at 4°C

5.

Save the following PRB_REM program on the thermal cycler:

•

Choose the preheat lid option and set to 100°C

•

Reaction volume is 30 µl

•

37°C for 15 minutes

•

70°C for 15 minutes

•

Hold at 4°C

Hybridize Probes

Hybridize Probes (Illumina Stranded Total RNA with Ribo-Zero Plus)

If using Stranded Total RNA with Ribo-Zero Plus Microbiome, continue to Hybridize Probes (Illumina Stranded Total RNA with Ribo-Zero Plus Microbiome).

1.

Using nuclease-free ultrapure water, dilute 1–1000 ng total RNA to a volume of 11 µl in each well of a new PCR plate.

2.

In a 1.7 ml tube on ice, combine exactly the following volumes to prepare Hybridize Probe Master Mix. Multiply each volume by the number of samples.

•

DB1 (3.6 µl)

•

DP1 (1.2 µl)

Volumes include reagent overage for accurate pipetting.

3.

Thoroughly pipette Hybridize Probe Master Mix to mix.

4.

Add 4 µl Hybridize Probe Master Mix to each well.

5.

Pipette 10 times, and then seal.

6.

Place on the preprogrammed thermal cycler and run the HYB_DP1 program.

Total program time is ~15 minutes and each well contains a volume of 15 µl.

7.

Continue to Deplete rRNA.

Hybridize Probes (Illumina Stranded Total RNA with Ribo-Zero Plus Microbiome)

1.

Using nuclease-free ultrapure water, dilute 25–500 ng total RNA to a volume of 10 µl in each well of a new PCR plate.

2.

In a 1.7 ml tube on ice, combine exactly the following volumes to prepare Hybridize Probe Master Mix. Multiply each volume by the number of samples.

•

DB1 (3.6 μl)

•

DP1 (1.2 μl)

•

DPM (1.2 μl)

Volumes include reagent overage for accurate pipetting.

3.

Thoroughly pipette Hybridize Probe Master Mix to mix.

4.

Add 5 μl Hybridize Probe Master Mix to each well.

5.

Pipette 10 times, and then seal.

6.

Place on the preprogrammed thermal cycler and run the HYB_DP1 program. Total program time is ~15 minutes and each well contains a volume of 15 μl.

7.

Continue to Deplete rRNA.

Deplete rRNA

1.

In a 1.7 ml tube on ice, combine exactly the following volumes to prepare rRNA Depletion Master Mix. Multiply each volume by the number of samples.

•

RDB (4.8 µl)

•

RDE (1.2 µl)

Volumes include reagent overage for accurate pipetting.

2.

Thoroughly pipette rRNA Depletion Master Mix to mix.

3.

Centrifuge the sealed PCR plate at 280 × g for 10 seconds.

4.

Add 5 µl rRNA Depletion Master Mix to each well.

5.

Pipette 10 times, and then seal.

6.

Place on the preprogrammed thermal cycler and run the RNA_DEP program.

Total program time is ~17 minutes and each well contains a volume of 20 µl.

Remove Probes

1.

In a 1.7 ml tube on ice, combine exactly the following volumes to prepare Probe Removal Master Mix. Multiply each volume by the number of samples.

•

PRB (7.7 µl)

•

PRE (3.3 µl)

Volumes include reagent overage for accurate pipetting.

2.

Thoroughly pipette Probe Removal Master Mix to mix.

3.

Centrifuge the sealed PCR plate at 280 × g for 10 seconds.

4.

Add 10 µl Probe Removal Master Mix to each well.

5.

Pipette 10 times, and then seal.

6.

Place on the preprogrammed thermal cycler and run the PRB_REM program.

Total program time is ~32 minutes and each well contains a volume of 30 µl.

Clean Up RNA

1.

Centrifuge the sealed PCR plate at 280 × g for 10 seconds.

2.

Vortex RNAClean XP to resuspend.

3.

Add 60 µl RNAClean XP to each well.

4.

Mix using either method:

•

Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.

•

Slowly pipette until the beads are resuspended.

5.

Incubate at room temperature for 5 minutes.

6.

Place on the magnetic stand and wait 5 minutes.

7.

Remove and discard 80 µl supernatant.

8.

Wash beads as follows.

a.

Keep on the magnetic stand and add 175 µl fresh 80% EtOH to each well.

b.

Wait 30 seconds.

c.

Remove and discard all supernatant from each well.

9.

Wash beads a second time.

10.

With a 20 µl pipette, remove all residual EtOH.

11.

Air-dry on the magnetic stand for 2 minutes. Do not overdry the beads.

12.

Remove from the magnetic stand.

13.

Add 10.5 µl ELB to each well.

14.

Mix using either method:

•

Seal the plate and centrifuge at 280 × g for 10 seconds, and then shake at 2200 rpm for 1 minute.

•

Slowly pipette until the beads are resuspended, and then seal.

15.

If shaking did not fully resuspend the beads, slowly pipette until the beads are resuspended, and then seal.

16.

Incubate at room temperature for 2 minutes.

17.

Centrifuge at 280 × g for 10 seconds.

18.

Place on the magnetic stand and wait 2 minutes.

19.

Transfer 8.5 µl supernatant to the corresponding well of a new PCR plate.

Small amounts of bead carryover do not affect performance.