rRNA depletion RNAseq protocol

Ambros Lab rRNA depletion RNAseq protocol Ye (Oscar) Duan

(Version 10.20.2020)

1. Annealing

In self-lid PCR tubes, make the following mixture:

Incubate at 95 °C for 2 min, then 65 °C for 30 min.

For first time user, we suggest sampling the RIN of the input total RNA by bioanalyzer. This step does not guarantee the quality of RNA after the depletion but can indicate the input quality.

2. Digestion

With the tubes in the thermocycler or heating block, make the following reaction by adding the master-mix into each tube. Mix by pipetting up and down for 10 times. We suggest preheating the master mix containing RNaseH at 65 °C for 1min before adding to the reaction. To reduce evaporation, the operations should be very fast, and the PCR tube lids should be open only once at a time.

Incubate at 65 °C for 40 min, then chill the tubes on ice for more than 2 min.

3. ASO removal

Mix the following mix:

Incubate at 37 °C for 25 min, then chill the tubes on ice for more than 2 min.

4. Purification

Purify and concentrate the RNA product using Zymo R1015/1016 as follow (volume for one sample): (1). Mix 105 ul RNA Binding Buffer with 105 ul ethanol.

(2). Add 200 ul adjusted RNA Binding Buffer to sample, mix by vortex. (3). Load mixture to Zymo-Spin Column and centrifuge for 30 sec.

1. Add 400 ul RNA Prep Buffer to the column and centrifuge for 30 sec. Discardflow-through.
2. Add 700 ul RNA Wash Buffer to the column and centrifuge for 30 sec. Discard flow-through (make sure ethanol has been added for the first-time use).
3. Add 400 ul RNA Wash Buffer to the column and centrifuge for 2 min. Transfer the column carefully into RNase-free tube.
4. Add 10 ul H₂O to the column matrix, incubate at RT for 30 sec. Centrifuge for 30 sec.

5. Quantification.

Determine the concentration the RNA using Qubit HS RNA assay following instruction. Use 3-5 µl RNA for assay.

6. Library prep.

Prep library using NEB E7775 and E7335/E7500 using the following parameters (for 0.5X reaction):

Input: 5-10 ng total rRNA depleted in Step 5. (2.5 ul according to this protocol, note that 5-10 ng input in our protocol (0.5X) equals to 10-20 ng in the NEB manual (1X)

Fragmentation: 15 min.

Adaptor dilution: 1:5.

Amplifying cycles: 8-9.

7. Library quantification.

Size distribution: Fragment analyzer (High Sensitivity dsDNA)

Concentration: KAPA quantification kit (at least 1:2000 dilution) or NEB E7630.

8. Buffers and Reagents:

ASO: 120mer ASO antisense to 26S, 18S, 5.8S (49X), stock has 2 µM for each (total 98 µM).

60mer ASO antisense to 5S (3X), 100 µM for each. 60mer SRPR antisense (9X), 100 µM for each.

  These ASOs are pooled in a 0.8 µM pool. We recommend GENEWIZ to synthesize the oligos.

5X annealing buffer: 50 mM Tris-HCl (pH=8.0), 0.5M NaCl, 5 mM EDTA

MCLab: HTRH-100

NEB: M0314S E7775S E7500S E7335S E7630(optional)

ThermoFisher: AM2238 Q32852 Zymo Research: R1015/R1016 Roche: KK4824

Fragment analyzer:

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