Preparation and harvesting of 4SU incorporated mouse tissues
1. For tagged proteins expressed by exogenous delivery, start by administration of the expression system into the mice to achieve the needed expression value by the day of 4SU administration. Routinely, we use adenovirus-mediated gene delivery to specifically target hepatocytes. Therefore, we intravenously inject 3x109 PFU in a total volume of 200 μL PBS into mice, 7-10 days prior to the experiment.
2. Prepare a fresh solution of 4SU (NOTE 1) in PBS (injection grade) corresponding to a dose of 780 mg/kg per mouse in a total volume of 200 μL. After the 4SU has dissolved, sterile filter the solution and store in the dark at 4 °C. Use this solution within the next 24-48 hours.
3. Administer 200 μL of the 4SU solution, that has been warmed up to room temperature, by intraperitoneal injections (NOTE 2). The number of animals depends on the tissue specific RNA binding protein expression, its target abundance, crosslink efficiency and tissue of interest. We routinely use 5 animals and combine the organs to bigger batches.
4. Inject the solution every 2.5 hours (NOTE 3), over the time-course of 12.5h. After the last injection wait 2.5 hours before scarifying the mice, so that the mice are exposed to 4SU for a total of 15 hours (NOTE 4).
5. Euthanize the mice using carbon dioxide (CO2) until breathing stops. Next, transcardially perfuse the mice with ice-cold PBS. Excise the organs rapidly and immediately snap-freeze the tissue in liquid nitrogen. If studying organs with a high content of ribonucleases and proteinases, e.g., the pancreas, start dissecting these organs first.
6. Once frozen in liquid nitrogen, the tissues can be stored indefinite or at least 12 months at -80°C.
Tissue grinding and UV365-Crosslinking
1. Pre-cool the tissue grinder , spatula, razorblade and crosslinking-pad with liquid nitrogen.
2. Remove the tissue from -80 °C and cool down further with liquid nitrogen.
3. Once the temperature is reached, add the tissue into the grinder and produce a fine powder with several strokes and by mixing the powder with the spatula between the strokes. It can be necessary to cool down the tissue grinder between these steps. Repeat the steps until a homogeneous fine powder is reached.
4. Next, spread the powder as a fine layer onto the liquid nitrogen cool-pad and crosslink it four times with 0.300 J/cm-2 at 365 nm (NOTE 5). Mix the powder with the help of a razorblade between each irradiation and make sure the cooling is secured.
5. Collect the crosslinked tissue powder into a pre-cooled 50 mL falcon and snap freeze it in liquid nitrogen. The crosslinked powder can be stored indefinite or at least 12 months at -80 °C.
3.1 Tissue lysis and RNase treatment
1. Homogenize the tissue powder (NOTE 6) in a minimum of ice-cold high percentage NP40 buffer using a glass douncer with 20 strokes in an ice bath.
2. Incubate the lysate on a rotation wheel at 4 °C for 1-2 hours (start preparing the beads during this incubation time).
3. After incubation, sonicate the lysate in a Bioruptor-Plus with the setting “low” for 5 cycles 30sec on/off time (NOTE 7).
4. Dilute the lysate with dilution buffer to adjust the NP40 concentration according to the antibody-antigen recognition requirements and to reach a tissue lysis buffer ratio of 1:10 (NOTE 8).
5. Clear the lysate by centrifugation at 13,000 g at 4 °C for 30 min and filter through a 0.45 μm membrane filter (low protein binding filter). If the tissue has a high amount of fat, it can be necessary to remove the fat layer before filtration.
6. For an efficient lysis and recovery of RNAs in the fragment size of 20-40 nt, add the upfront determined concentration of RNaseT1 to the lysate (NOTE 9).
3.1 Bead preparation
1. Prepare a fresh tube with 5 mg protein G magnetic beads per mL lysate.
2. Place the tube on the magnetic rack to separate the beads from the storage solution and wash the beads three times with bead wash buffer. Do not let the beads get dry at any point.
3. After the final wash remove the bead wash buffer and add 10 μg of the antibody adjusted to the original bead volume with PBS-Tween (NOTE 10).
4. Incubate the beads by rotation at room temperature for 2 hours.
5. After incubation wash the beads twice with ice-cold NP40 lysis buffer that has the same final NP40 concentration as described in 3.3 point 4.
1. Remove the buffer from the beads prepared in 3.4 and directly add the lysate prepared in step 3.3.
2. Incubate the samples by rotating them at 4 °C overnight.
3. Collect the beads with the magnetic stand and discard or use the supernatant for subsequent immunoprecipitations with other antibodies, keeping in mind the long ribonuclease T1 incubation. If performing subsequent immunoprecipitations, add additional ribonuclease and proteinase inhibitors to the lysate. It can be necessary to also stop the ribonuclease T1 digestion at this point, by choosing an appropriate inhibitor.
4. Wash the beads three times with IP wash buffer (NOTE 11).
5. Wash the beads twice in NEB3 buffer and resuspend the beads in 1 volume of NEB3.
6. Add calf intestinal alkaline phosphatase (10 U/μl) to a final concentration of 0.5 U/μl, and incubate the suspension for 30 min at 37°C with shaking the beads every 2 min for 15 sec at 1200 rpm.
7. Wash the beads twice in phosphatase wash buffer.
8. Wash the beads twice in PNK buffer without DTT and resuspend the beads in 1 volume of PNK buffer with DTT and immediately continue with the radiolabeling reaction as described in the original protocol (starting from step 3.3.6) Garcia et al.1 (NOTE 12).
All the following steps; recovery of RNA-RBP complex from nitrocellulose membrane, proteinase K digestion, RNA fragment size selection, adapter ligation, cDNA library preparation, PCR amplification, and computational data analysis are done according to the previously published protocol Garcia et al.1.
1. Before using 4SU for in vivo application a quality control should be performed to ensure purity of the product. Bigger batches of 4SU are therefore subjected to 1H-NMR and 13C-NMR 16. Degradation products or impurities should be removed by recrystallization or column chromatography with HPLC.
2. Make sure that this experimental protocol is approved by the animal use & care committees and in compliance with the guidelines according your institution and country location.
3. We routinely performed our experiments with C57BL/6 mice; however, it can be necessary to measure the clearance of 4SU in the blood (extended methods: Measurement of plasma 4SU pharmacokinetics via UV-VIS) to adjust the injection regimen for other mouse strains, with successively determination of incorporation rates of 4SU in the total RNA of the tissue of interest (extended methods: Measurement of 4SU incorporation rates in whole tissue total).
4. This injection regimen will capture the majority of transcript, that have a half-life which falls into that timeframe. It can be useful to adjust the time for transcripts with longer or shorter half-lives. For short-lived transcripts, reduce the time between the last injection and time of sacrifice.
5. For each new tissue and RBP the crosslinking should be optimized. Therefore, crosslink the tissue with increasing energy and choose the energy setting right under the crosslink saturation point. Prolonged exposure to UV light will produce undesirable RNA phosphodiester backbone breaks or RNA-RNA inter- and intrastrand crosslinks.
6. The starting amount of tissue powder depends on the RBP protein and its crosslinking ability and needs to be determined experimentally. For large scale Tial1 viP-CLIP in liver we usually start with 3 g.
7. For an efficient sonication, do not exceed the maximum volume of lysate per tube as indicated in the instruction manual. E.g., 1.5 mL Eppendorf tubes shouldn’t be filled up with more than 300 μL.
8. A high NP40 concentration of >1% is necessary to efficiently capture nuclear interaction e.g., for RBPs with intronic binding affinity. However, the high concentration of detergent can be detrimental for the antibody-antigen recognition and needs to be determined before proceeding with the viP-CLIP protocol.
9. Omitting the ribonuclease treatment during the IP largely captures RBPs that are less bound to RNA or with a bias to shorter RNAs, as indicated by the same amount of immunoprecipitated proteins. However, the amount of crosslinked RNA decreases as the IP of large RBP-RNA complexes is difficult to immunoprecipitated. Therefore, the ribonuclease treatment should always be performed during the incubation with the beads.
10. Protein G Dynabeads: check the binding affinity and capacity of the beads to the specific antibody used and change the ratio of bead to antibody according to the manufacturer notes. We routinely couple 10 μg antibody to 5 mg protein G magnetic beads.
11. Depending on the magnetic-beads and conjugated antibody, the ionic strength of the salt concentration can interfere with the binding ability of the antibody. Therefore, the salt concentration of the IP-wash and the high-salt wash buffer needs to be determined before conducting the experiment.
12. For viP-CLIP we routinely blot the RBP-RNA complexes to nitrocellulose. This helps to detect RBPs with lower crosslinking signals and removes non-crosslinked background RNAs that migrate together with the RBP-RNA in the SDS-PAGE gel.