TITLE: DAT-TRAP Protocol
AUTHORS: Peter Kilfeather

[DESCRIPTION]
This protocol describes the capture of eGFP-L10a-tagged ribosomes and mRNA from DAT-expressing cells in mouse ventral midbrain.

[GUIDELINES]
Prepare all reagents under RNAse-free conditions, preferably with the use of a PCR hood.

[STEPS]
SECTION: Tissue Collection
2. Prepare all dissection instruments and collection tubes on ice. Set a refrigerated centrifuge to 4 °C. Be prepared to work swiftly, to minimise changes in translation occurring after death. Collection materials should be prepared in an RNase-free manner, to minimise the risk of sample degradation.
SECTION: Tissue Collection
3. Cull the mouse by cervical dislocation. Extract the brain and perform rapid chilling by submersion in Dissection Buffer. Place the brain into a matrix or onto a flat surface for sectioning. 
SECTION: Tissue Collection
4. From each section, dissect the target brain region. Immediately homogenise dissected tissue in Tissue Lysis Buffer using a dounce homogeniser. The volume of tissue lysis buffer used should be decided in optimisation experiments and should scale with the total mass of tissue dissected. For information on choosing an appropriate volume, see Dougherty et al., 2010.
 

To ensure consistent and thorough disruption, use a fixed number of strokes for every sample (e.g 20, 30) and select a pestle that provides minimal clearance. Avoid foaming by keeping the pestle below the surface of the buffer at all times.

Note: Tissue can be snap-frozen immediately after dissection, to facilitate collection of large numbers of samples. Alternatively, homogenized contents can be stored on ice while additional samples are collected. Tissue Lysis Buffer contains cycloheximide to stall translation.
SECTION: Immunoprecipitation
5. Transfer each lysate into ice-cold Eppendorf tubes and centrifuge at 2,000 x g at 4 °C for 10 minutes. Carefully transfer the supernatant to a new tube.
SECTION: Immunoprecipitation
6. To the supernatant, add 1/8th volume of 300 mM DHPC and 1/8th volume of 10 % NP-40. Mix the solutions by inversion. Hold the mixtures on ice for 5 minutes before centrifugation at 20,000 x g at 4 °C for 10 minutes. Carefully transfer the supernatant to a new tube.
SECTION: Immunoprecipitation
7. Transfer 50 ul of lysate into a separate tube to be used as a paired 'Input' sample. To ensure the same conditions are kept, hold this sample at 4 °C until the 'IP' sample is processed for RNA extraction the following day.
SECTION: Immunoprecipitation
8. Add titrated volume of anti-eGFP-coated paramagnetic bead matrix to the 'IP' sample. Rotate overnight at 4°C.
SECTION: Matrix preparation
1. Prepare anti-GFP-coated paramagnetic bead matrix according to Box 1 of Heiman et al., 2014.
 
The quantity of beads/antibody must be titrated according to the eGFP-content of the sample of interest. See the Supplementary text of Dougherty et al., 2010 further information.

 

SECTION: Washing and extraction
9. On day 2, proceed to washing the bead matrix: Place each IP sample on a magnetic rack to pellet beads on the sidewall. Aspirate and discard all supernatant. Resuspend the bead matrix in 1 mL of ice-cold High Salt buffer and dispense into a fresh tube.
SECTION: Washing and extraction
10. Incubate for 5 minutes on ice, repeat pelleting, resuspension and transfer to a fresh tube. Perform this washing step 6 times in total.
SECTION: Washing and extraction
11. After the final wash, pellet the bead matrix using a magnet rack, remove the supernatant, warm the tube to room temperature and resuspend in 100 µL of room temperature Buffer RLT-Plus with 1 % β-mercaptoethanol. Vortex vigorously and incubate for 10 minutes.
SECTION: Washing and extraction
12. Pellet the bead matrix using a magnetic rack and transfer the supernatant to a Qiagen RNEasy Micro collection column (Qiagen, #74034). Follow manufacturer's instructions for RNA extraction. Use 14 µL of nuclease-free water for RNA elution and divide the elute into 2 µL and 10 µL (allowing for 2 µL loss) volumes. Store the 10 µL volume immediately at -80 °C.
13. Hold the 2 µL volume at 4 °C and proceed to RNA yield quantification using the Quant-it™ RiboGreen RNA Assay Kit (ThermoFisher #R11490). Measure RNA integrity using the Agilent 2100 RNA Pico BioAnalyzer.