Stereotactic injection of RiboTag virus in the brain
Skip this section if using an alternative method to express RiboTag in the cells of interest (e.g.: generation of a specific transgenic mouse).
Different viral preparations might be required for specific cell types. In this specific case, a combination of cre-dependent viral construct (e.g: AAV9-EF1a-DIO-Rpl22l1-Myc-DDK-2A-tdTomato-WPRE) and mice expressing cre under the DAT promoter (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J) were used to achieve expression in midbrain dopaminergic neurons.
- Anesthesia machine (Smiths Medical) with connector tubing, induction chamber and filter canisters for isoflurane waste
- Stereotaxic surgery frame and scope (David Kopf Instruments)
- Sterile surgery tools (forceps, fine scissors, needle holder as needed)
- Heating pad and temperature probe
- Non-steroidal analgesic (e.g. Metacam)
- Antiseptics: povidone-iodine swabs and 70% ethanol swabs
- Scale to measure the weight of the mouse
- Drill with foot petal and sterilized drill bit
- EMLA cream or bupivacaine line block
- Glass micropipettes (Drummond Scientific) pulled with P-97 glass puller (Sutter Instruments). It is recommended to add some volumetric references on the pipettes based on their specifics.
- Post-surgery care: clean empty mouse cage on heating pad for recovery; clean mouse cage with extra gel food for post-surgery holding.
Depending on the biosafety level (BSL) recommended for type of virus injected, an appropriate biosafety cabinet might be required.
- Anesthetic (ketamine 50 mg/Kg and xylazine 4.5 mg/Kg – varies according to institutional protocols)
- Vibratome (VTS1200S Leica microsystems) with removable ice tray and cutting chamber and vibro-check tool
- 2 glass petri dishes (one of the two can be substituted with a medium weighing boat)
- 1 circular filter paper
- Peristaltic pump (Gilson) with tubing and connectors
- Blood-gas mixture (95% O2, 5% CO2) tank connected to bubblers.
- Dissection tools (scissors, fine scissors, spring scissors, tweezers, spatula, according to preferences)
- Double-edged razor blades
- Single-edge razor blades
- artificial cerebro-spinal fluid (aCSF), pre-chilled
- Freshly made “slicing solution”: modified aCSF.
- Pre-frozen slicing solution
- Perfusion needle (preferred: 27 gauge ½ inch)
- Holding chamber for slices, filled with pre-chilled aCSF and kept in ice
- Pre-solidified 2% agarose
- Plastic or glass transfer pipette
- Petri dishes for dissection with a small volume of ice cold aCSF (recommended: use petri dishes of different sizes and place the petri dish with aCSF inside the larger one, filling the space between with ice. This will help keeping the dissection solution cold).
- Dry ice or liquid nitrogen
- 1.5ml Eppendorf tubes to collect samples
- Lab gown/disposable gown
- Examination gloves (cut-resistant gloves are recommended)
- Cryo-gloves to handle dry-ice/liquid nitrogen
Different types of aCSF are adopted by different groups and are optimized for different for different preparations. For tissue dissection, it is recommended to use the solution normally used for experimental purposes, but pre-chilled.
The aCSF adopted for experiments on SNc neurons has the following composition: 135.75 mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 25 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, 3.5 mM glucose.
The slicing solution for SNc neurons has the following composition: 49.14 mM NaCl, 2.5 mM KCl, 1.43 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose, 99.32 mM sucrose, 10 mM MgCl2 and 0.5 mM CaCl2.
All the solutions are continuously bubbled with blood-gas mixture before and during the procedure. This will maintain the proper oxygenation and pH.
Immunoprecipitation and RNA extraction for RNAseq analysis
- Magnet (Dyna-Mag2, Thermo Fisher Scientific)
- Refrigerated minicentrifuge (Beckman Coulter 20R)
- RNase-free 1.5-ml microcentrifuge tubes (Axygen)
- RNase-free filtered tips
- RNase-free pestles for 1.5-ml microcentrifuge tubes (USA Scientific)
- Tube rotator (Thermo Fisher Scientific)
- Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific)
- Anti-FlagM2 Magnetic Beads (Sigma-Aldrich)
- SUPERase In RNase Inhibitor (Thermo Fisher Scientific)
- Protease inhibitors (Sigma-Aldrich)
- Cyclohexamide (Sigma-Aldrich)
- Nonident P-40 Substitute (Sigma-Aldrich)
- UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher Scientific)
- Ethanol (Sigma-Aldrich)
- RNAeasy Micro kit (Qiagen)
- RNaseZap (Thermo Fisher Scientific)
Tissue homogenization buffer has the following composition: 50mM Tris-HCl (pH 7.4), 125 mM KCl, 12 mM MgCl2, 1% NP-40, 1mM DTT, 1X Protease inhibitors, 200 units/ml RNase inhibitor, 100ug/ml cycloheximide.
Washing buffer has the following composition: 50mM Tris-HCl (pH 7.4), 325mM KCl, 12 mM MgCl2, 1% NP-40, 1mM DTT, 100ug/ml cycloheximide.