Translatome purification with RiboTag

Translatome purification with RiboTag

This polysome immunoprecipitation (IP) procedure was used i.a. in (Kaczmarczyk et al., 2021) and in (Kaczmarczyk et al., 2019) (the latter with modifications allowing co-purification of Ago2-bound miRNAs).

The procedure is based on the RiboTagpaper by Sanz et al. (Sanz et al., 2009),but contains important modifications, including using a different antibody for IP.

General remarks

• Typically, brain hemispheres are flash frozen in separate tubes (we use sealed cryo-vials to minimize drying and oxidation of samples over long-term storage at -80°C), which are then thawed and processed when convenient, but ideally without excessive delays. Flash freezing on dry ice is sufficient. We often use metal cooling/heating blocks cooled on dry ice to ensure reproducible freezing speeds for all specimens.
• Do not store brain samples at temperatures higher than -80°C.
• We recommend storing all samples to be used for quantitative analysis for approximately the same amount of time to avoid batch effects due to unequal storage duration.
• All reagents used for RNA work should be free of RNase contamination and RNA techniques should be always applied.
• Low binding, nuclease-free tubes should be used during RiboTag IP procedure.
• Throughout RiboTag IP procedure, samples should be always kept on ice. All incubations (antibody binding, washing steps, etc.) are done at 4°C.
• The amount of purifiedRNA depends on the volumeand concentration of Supernatant 1 (S1) and on the fraction of positive (i.e., expressing RiboTag) cells in the tissue (more abundant cell populations will yield more material and vice versa).
• We routinely use 200-350 μl of S1. For larger cell populations, amounts of S1 and beads may be scaled down, if desired. On the other hand, due to unknown reasons, scaling up (i.e., using larger volumes of S1 per IP) did not result in expected (proportional) increases in the final RNA yields.
• Before starting, cool down the centrifuge and prepare all the neededbuffers with freshadditives.
• Final RNA elution step may be done with RNAse-free TE buffer insteadof water in case long-term storage of RNA is envisioned.
   

Buffers

Polysome Buffer (PSB)

Tris, pH 7.5                                50 mM

KCl                                            100 mM

MgCl₂                                       12 mM

Nonidet P-40                            1%

(+) To an aliquot of this buffer just prior to use add (dilution of stock solution provided in brackets):

DTT                                            1mM (1:250)

RiboLock                                    2.5 µl/ml = 100U/ml

Cyclohexamide                          100 µg/ml (1:100)

SigmaFast +EDTA                      1-2x (1:5-1:10)

High Salt Buffer (HSB)

Tris, pH 7.5                                50 mM

KCl                                             300 mM

MgCl₂                                        12 mM

Nonidet P-40                             1%

(+) To an aliquot of this buffer just prior to use add (dilution of stock provided in brackets):

DTT                                            1mM (1:250)

RiboLock                                    2.5 µl/ml = 100U/ml

Cyclohexamide                          100 µg/ml (1:100)

SigmaFast +EDTA                       0.5x (1:20)

Extra high salt buffer (EHSB)

Tris, pH 7.5                                 50 mM

KCl                                              300 mM

NaCl                                           300 mM

MgCl₂                                        12 mM

Nonidet P-40                             1%

(+) To an aliquot of this buffer just prior to use add (dilution of stock provided in brackets):

DTT                                             1mM (1:250)

RiboLock                                    2.5 µl/ml = 100U/ml

Cyclohexamide                          100 µg/ml (1:100)

SigmaFast +EDTA                      0.5x (1:20)

Preparation of 10% brain homogenate

1. Weigh the brain tissue in a cryo-vial (be quick not to let the tissue thaw at this point).
   Tip: a clean empty cryo-vial can be used to TARE the balance as the error is typically negligible.
2. Aliquot PSB+ into a 5 ml Teflon-glass (Potter-Elvehjem) homogenizer and chill on ice appropriate volume of PSB+ (for 10% homogenate: 0.1 ml PSB/0.01 g tissue, e.g., 0.22 g brain requires 2.2 ml buffer). Do not make S1 more than 10% concentrated!
3. Drop the tissue piece into the homogenizer filled with PSB+.
   Tip: deep-frozen brain stuck inside the tube can be quickly dislodged (while still frozen) through hitting the tube against a hard surface a couple of times.
4. Prepare the homogenate using a motorizedhomogenizer (e.g., HeidolphHei-Torque) set to 450 rpm. Move the pestle in the homogenizer up and down (~0.5-1 cycle/s, ~20-30x, which amounts to ~1 min of total homogenization time) without forming excessive air bubbles. Keep the homogenizer in a beaker or falcon filled with ice to keep the samples cold.
5. Centrifuge the homogenate (10.000 g / 10 min / 4°C) to prepare S1. During centrifugation, aliquot 700 µl of Qiazol into 1.5 ml tube (this will be used for isolation of total RNA from S1).
   Note: Pellet 1 (P1) containing total brain nuclei can be used for optional analyses if desired (e.g., DNA analysis).
6. Transfer S1 to a clean tube (2 x 1.5 ml Eppendorf or 15 ml falcon tube).
7. Take 30-60 µl of S1 and mix with prepared Qiazol aliquot. This is the total RNA sample from S1 and can be stored frozen until RNA purification.
8. Proceed immediately to immunoprecipitations.

RNA immunoprecipitation (IP)

1. Pre-clearing
   1.1 Wash PGDB (25 µl beads suspension per 300 µl of S1) 2x with PBS, 1x with PSB. Add S1 to equilibrated beads (300 µl S1 per IP).
   1.2 Place the mixture on a rotator for gentle mixing.
   Tip: optionally, beads for pre-clearing can be used several times, bound materialfrom the beads might to be eluted by 50 mM glycine (pH 2.8) on the Eppendorf Thermomixer (700 rpm, 30 min), the beads have to be washed twicewith PBS-T and stored in PBS-T, and then just before using washed again with PBS-T at least one time.
   1.3. Put the pre-clearing mixture on a magnetic rack; let the beads adhere to the wall and then to proceed to parallel or sequential procedure variant.
2. Mix 300 µl of pre-cleared S1 with 12 µl (6 µg) of anti-HA mAb 12CA5 and place the tube on a rotator.
3. After 60 min transfer the mixture to a tube containing PGDB (equilibrated in PSB) and incubate for additional 90 min.
4. After the incubation, place the samples on a magnetic rack and allow the beads to completely adhere to the tube wall.
   Note: At this point unbound fraction 1 (UF1) for western blot can be collected by mixing a small aliquot of sample with an equal volume of SDS-PAGE sample buffer (e.g., 2x LDS NuPage buffer).
   
   Table 1. Summary amounts of beads and antibodies used in RiboTag IPs

Washing magnetic beads and RNA purification

1. Wash the beads 3 times with HSB+ and then 3 additional times with EHSB+ (600 μl per wash). Each wash should be done carefully not to lose any beads and to collect as much liquid aspossible. Do not vortex the beads. Instead, let them be mixed only through rotation on the rotator. Leaving the beads on a rotator for 2-5 min for each wash helps to reduce background.
   Optional: after the last wash and before removing the buffer, remove 5-10% of the bead suspension for western blot¹.
   ¹Western blot samples from the beads can be prepared the sample by removing the buffer on a magnet and then adding SDS-PAGE reducing sample buffer. These can be frozen immediately and denatured prior to running on gel. Use a magnet to prevent aspiration of the beads while loading the samples.
2. Add 700 μl QIAzol to the beads.
3. Shake on a rotator/thermomixer (max speed, RT) for 5-10min.
   Note: this is a convenient pause point and the samples in Qiazol can be stored frozen at -80°C.
   Note: the remainder of the procedure is based on Qiagen miRNAeasy micro kit protocol with minor modifications.
4. Add 140 μl chloroform and cap tube securely. Shake and/or vortex vigorously for > 15 s.
5. Optional: transfer everything to 2 ml Phase Lock heavy gel tube (previously spun down briefly at max speed to pellet the gel).
6. Incubate at room temperature (RT) for 2–3 min, then centrifuge for 15 min at 12.000 g / 4°C.
7. Transfer the upper aqueous phase to a new tube (for PGDB this is 420-440 µl). Using Phase lock beads helps to remove whole upper phase, which might be important for low abundant samples. Avoid transferring any interphase. Add 1.5 volumes (usually 630 μl per 420 μl aqueous phase) of 100% ethanol and mix thoroughly by pipetting.
8. Pipet up to 700 μl sample into an RNeasyMinElute spin column (these are stored at 4°C) in a 2 ml collection tube. Close the lid and centrifuge (9000 g / 15 s / RT). Discard the flow- through.
9. Repeat step 7 using the remainder of the sample.
10. Add 700 μl of Buffer RWT to the spin column. Close the lid and centrifuge (9000 g / 15 s / RT). Discard the flow-through.
11. Add 500 μl of Buffer RPE onto the spin column. Close the lid and centrifuge (9000 g / 15 s / RT). Discard the flow-through.
12. Add 500 μl of 80% ethanol to the spin column. Close the lid, and centrifuge (9000 g / 1 min / RT). Discard the flow-through and the collection tube.
13. Place the spin column in a new 2 ml collection tube (supplied with the kit). Centrifuge at >13.000 g full speed for 5 min to dry the membrane. Discard the flow-through and the collection tube.
14. After centrifugation, remove the spin column carefully and place in a new 1.5 ml collection tube.
15. Add 14 μl RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥ 10.000g to elute the RNA. Then (option 1) apply the same eluate onto the filter again, let it sit for 1 min @ RT and spin again. To maximize yield (option 2), perform the second elution with new aliquot of water instead.
16. Freeze the samples and -80°C or keep on ice if used immediately for downstream applications.

Buffers and stock solutions

All solutions and buffers for RNA work are made using RNAse free water. Stock solutions should either be purchased as RNase free or preparedusing certified reagentsin RNAse-free environment and sterile-filtered.

Other materials

Motorized drill for homogenization pestle with adjustable speed (e.g., Heidolph Hei-Torque) Agilent Bioanalyzer (or equivalent device)

Nanophotometer

MacsMIX Tube Rotator (Miltenyi, cat. 130-090-753)

Miltenyi MACS rotator is very convenient because is both AC/DC and battery-powered and has a removable tube holder which may functionas a rack in which the tubes can be conveniently transferred between rotator and workbench.

RNA Bioanalyzer Kit Pico (Nano sufficient if expected yields are large) miRNeasy micro RNA purification kit (Qiagen, cat. 217084)

Protein G Dynabeads, PGDB (ThermoFisher, cat. 10003D) RiboLock RNAse inhibitor (ThermoFisher, cat. FEREO0382) DynaMag2 (ThermoFisher, cat. 12321D)

PhaseLock heavy gel 2ml tubes (5-Prime, cat. 2302830) Eppendorf Thermomixer (Eppendorf, cat. 5350 000.013)

Sigma Fast protease inhibitor tablets with EDTA (Sigma, cat. S8820) Mouse-anti-HA clone HA7 (Sigma, cat. H3663) (for Western blot)

Mouse anti-HA clone 12CA5 (Roche, cat. 11583816001) (for RiboTag IP)

References

Kaczmarczyk, L., Bansal, V., Rajput, A., Rahman, R.U., Krzyzak, W., Degen, J., Poll, S., Fuhrmann, M., Bonn, S., and Jackson, W.S. (2019). Tagger-A Swiss army knife for multiomics to dissect cell type-specific mechanisms of gene expression in mice. PLoS Biol 17, e3000374.

Kaczmarczyk, L., Reichenbach, N., Blank, N., Jonson, M., Dittrich, L., Petzold, G.C., and Jackson,

W.S. (2021). Slc1a3-2A-CreERT2 mice reveal unique features of Bergmann glia and augment a growing collection of Cre drivers and effectors in the 129S4 genetic background Sci Rep 11, 5412.

Sanz, E., Yang, L., Su, T., Morris, D.R., McKnight, G.S., and Amieux, P.S. (2009). Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A 106, 13939-13944.