3 Methods
Recommended total RNA input amount: 50 ng to 1.5 μg, RNA
integrity number (RIN) > 7. For more details on platform selection, see Subheading 1.1 in Introduction.
3.1 Ribosomal RNA
Depletion
We outline below a protocol for rRNA depletion with siTOOLs
Biotech’s riboPOOL, which we have found to achieve high rRNA
depletion in M. tuberculosis.
1. Centrifuge tube containing riboPOOL at 11,000 Â g for 30 s
before opening.
2. Resuspend riboPOOL in nuclease-free water provided to a final
concentration of 100 μM (e.g., 50 μL for 5 nmol).
3. To 14 μL of RNA sample, add:
(a) 1 μL of resuspended riboPOOL.
(b) 5 μL of Hybridization Buffer (if sample volume is >14 μL,
adjust HB volume according to 0.25Â total volume. Total
volume, however, should not exceed 40 μL). The resuspended riboPOOL volume should not change.
(c) RNase inhibitor (optional)—Follow manufacturer’s
instructions for volume required and ensure enzyme is
active at 68
C. RNase inhibitor may also be introduced
during bead preparation.
4. Vortex well and spin down droplets.
5. Incubate at 68
C for 10 min to denature RNA.
6. Allow to cool slowly from 68
C to 37
C for optimal hybridization. To do this using temperature-controlled ramping, cool
at 3
C/min (recommended). Alternatively, shut off the heating block and let temperature fall naturally to 37
C.
7. Resuspend the Dynabeads by carefully vortexing tube at
medium speed at room temperature.
8. Transfer 160 μL of Dynabeads bead suspension (10 mg/mL)
per sample into a fresh tube. To prepare multiple samples,
aliquot bead suspension for up to 6 samples (i.e., 960 μL) in
a single tube.
9. Place tube on magnetic rack and wait for 1 min. Beads may
stick to sides of tube making solution and appear brown, but
aspirated solution should be clear.
10. Briefly centrifuge the tube containing ~20 μL of hybridized
riboPOOL and total RNA (from step 6) to spin down
droplets.
11. Combine 80 μL of prepared beads (from step 9) with ~20 μL
of the hybridized riboPOOL-RNA solution. Agitate the tube
to resuspend well.
490
Shuyi Ma et al.
Recommended total RNA input amount: 50 ng to 1.5 μg, RNA
integrity number (RIN) > 7. For more details on platform selection, see Subheading 1.1 in Introduction.
3.1 Ribosomal RNA
Depletion
We outline below a protocol for rRNA depletion with siTOOLs
Biotech’s riboPOOL, which we have found to achieve high rRNA
depletion in M. tuberculosis.
1. Centrifuge tube containing riboPOOL at 11,000 Â g for 30 s
before opening.
2. Resuspend riboPOOL in nuclease-free water provided to a final
concentration of 100 μM (e.g., 50 μL for 5 nmol).
3. To 14 μL of RNA sample, add:
(a) 1 μL of resuspended riboPOOL.
(b) 5 μL of Hybridization Buffer (if sample volume is >14 μL,
adjust HB volume according to 0.25Â total volume. Total
volume, however, should not exceed 40 μL). The resuspended riboPOOL volume should not change.
(c) RNase inhibitor (optional)—Follow manufacturer’s
instructions for volume required and ensure enzyme is
active at 68
C. RNase inhibitor may also be introduced
during bead preparation.
4. Vortex well and spin down droplets.
5. Incubate at 68
C for 10 min to denature RNA.
6. Allow to cool slowly from 68
C to 37
C for optimal hybridization. To do this using temperature-controlled ramping, cool
at 3
C/min (recommended). Alternatively, shut off the heating block and let temperature fall naturally to 37
C.
7. Resuspend the Dynabeads by carefully vortexing tube at
medium speed at room temperature.
8. Transfer 160 μL of Dynabeads bead suspension (10 mg/mL)
per sample into a fresh tube. To prepare multiple samples,
aliquot bead suspension for up to 6 samples (i.e., 960 μL) in
a single tube.
9. Place tube on magnetic rack and wait for 1 min. Beads may
stick to sides of tube making solution and appear brown, but
aspirated solution should be clear.
10. Briefly centrifuge the tube containing ~20 μL of hybridized
riboPOOL and total RNA (from step 6) to spin down
droplets.
11. Combine 80 μL of prepared beads (from step 9) with ~20 μL
of the hybridized riboPOOL-RNA solution. Agitate the tube
to resuspend well.
490
Shuyi Ma et al.