Jan 10, 2024

Public workspaceDAT-TRAP Protocol V.2

DOI
dx.doi.org/10.17504/protocols.io.6qpvr4eo2gmk/v2
  • 1University of Oxford
Peter Kilfeather
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr4eo2gmk/v2
Protocol Citation: Peter Kilfeather 2024. DAT-TRAP Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4eo2gmk/v2Version created by Peter Kilfeather
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 10, 2024
Last Modified: January 10, 2024
Protocol Integer ID: 93238
Abstract
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.
Materials

  • Tissue-lysis buffer, Low-salt buffer and High-salt buffer made according to the Reagent Setup section of Heiman et al., 2014:

  • Tissue-lysis buffer: Mix 20 mM HEPES KOH (pH 7.4), 150 mM KCl and 10 mM MgCl2in RNase-free water. Store it at 4 °C for several months. Add EDTA-free protease inhibitors, 0.5 mM DTT, 100 μg/ml cycloheximide and 10 μl/ml rRNasin and Superasin to an aliquot immediately before use.

  • Low-salt buffer: Mix 20 mM HEPES KOH (pH 7.3), 150 mM KCl, 10 mM MgCl2and 1% (vol/vol) NP-40 in RNase-free water. Store the buffer at 4 °C for up to several months. Add EDTA-free protease inhibitors (one mini tablet per 10 ml), 0.5 mM DTT, 100 μg/ml cycloheximide and 10 μl/ml rRNasin and Superasin to an aliquot immediately before use.

  • High-salt buffer: Mix 20 mM HEPES KOH (pH 7.3), 350 mM KCl, 10 mM MgCl2and 1% (vol/vol) NP-40 in RNase-free water. Store it at 4 °C for up to several months. To an aliquot, add DTT to a final concentration of 0.5 mM and cycloheximide to a final concentration of 100 μg/ml immediately before use.

Reagent list
ABCD
ItemSpecific nameVendorCode
anti-eGFP antibody (clone 19C8)anti-eGFP antibody (clone 19C8)Memorial Sloan KetteringHeintz Lab TRAP anti-GFP 19C8
anti-eGFP antibody (clone 19F7)anti-eGFP antibody (clone 19F7)Memorial Sloan KetteringHeintz Lab TRAP anti-GFP 19F7
RNaseZapRNaseZap RNase Decontamination Wipes-100 sheetsLife TechnologiesAM9786
HEPESHEPES, 1 M, 100 mL, pH 7.3, RNase-freeFisher10041703
KClKCl, 2M, 100 mL, RNase-freeLife TechnologiesAM9640G
MgCl2MgCl2, 1M, 100 mL, RNase-freeLife TechnologiesAM9530G
RNase-free water
EDTA-free Protease InhibitorscOmplete, Mini, EDTA-free Protease ISigma11836170001
DTTDL-DTTSigmaD9779-1G
CycloheximideCycloheximide from Steptomyces griseus, 1gSigmaD769801G
rRNasinRecombinant RNasin Ribonuclease Inhibitor, 10,000uPromegaN2515
SuperasinSUPERase In RNase Inhibitor (20 U/uL)-10,000 unitsLife TechnologiesAM2696
HBSSHBSS (10X), calcium, magnesium, no phenol red-500Life Technologies14065056
GlucoseD-(+)-GLUCOSE BIOXTRASigmaG7528-250G
NaHCO3SODIUM BICARBONATE BIOXTRAS6297-250G
NP-40
Streptavidin DynabeadsDynabeads MyOne Streptavidin T1-2 mLLife Technologies65601
Streptavidin DynabeadsDynabeads MyOne Streptavidin T1-10 mLLife Technologies65602
NaOH
NaCl
BSA IgG-free, RNase-freeBovine Serum Albumin (IgG-Free, Protease-Free)Stratech Scientific001-000-162-JIR-50g
Proclin 300PROCLIN 300, 50MLSigma48912-U
DEPC waterAutoclave
Graeff forcepsSize 5 Graeff Forceps
Cutting Blades
Dounce homogenisersDOUNCE TISSUE GRINDER, ALL-GLASS, 2 MLSigmaD8938-1SET
1.5 mL DNA-LoBind Eppendorf TubesEppendorf® DNA LoBind tubesSigmaEP0030108051-250EA
Cervical dislocation tool
Surgical scissors
Fine scissors for skull
Spatula for brain
PBS
Wide orifice P200 tips
DHPC1,2-Diheptanoyl-sn-Glycero-3-Phosphocholine (DHPC) - Purity: ≥ 99% (by HPLC analysis) Generon (Non-catalogue)D607-250MG
B-Mercaptoethanol
70% Ethanol
80% Ethanol
RNeasy Micro ColumnsRNeasy Plus Micro Kit (50)Qiagen74034
DNA Eliminator Columns
Buffer RLT+
Buffer RW1
Buffer RPE
Elution water
Ribogreen KitQuant-iT RiboGreen RNA Assay Kit-1 kitLife TechnologiesR11490
Magnetic rackDynaMag-2ThermoFisher12321D
PCR Hood HEPA FilterHEPA Filter for 32 Laminar Flow PCR CabinetStarlabN3942-7000
PCR Hood HEPA Pre-FilterPre-Filters for 32 Laminar Flow PCR CabinetStarlabN3942-7500

Safety warnings
Attention
Toxicity: Contact your departmental safety office for specific advice about handling and safe disposal of cycloheximide.
Ethics statement
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC)
Matrix preparation
Matrix preparation
Prepare anti-GFP-coated paramagnetic bead matrix according to Box 1 of Heiman et al., 2014.
CITATION
Heiman M, Kulicke R, Fenster RJ, Greengard P, Heintz N (2014). Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).. Nature protocols.
LINK
https://doi.org/10.1038/nprot.2014.085
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.

CITATION
Dougherty JD, Schmidt EF, Nakajima M, Heintz N (2010). Analytical approaches to RNA profiling data for the identification of genes enriched in specific cells.. Nucleic acids research.
LINK
https://doi.org/10.1093/nar/gkq130

Tissue Collection
Tissue Collection
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.
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.
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.
CITATION
Dougherty JD, Schmidt EF, Nakajima M, Heintz N (2010). Analytical approaches to RNA profiling data for the identification of genes enriched in specific cells.. Nucleic acids research.
LINK
https://doi.org/10.1093/nar/gkq130

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.
Immunoprecipitation
Immunoprecipitation
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.
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.
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.
Add titrated volume of anti-eGFP-coated paramagnetic bead matrix to the 'IP' sample. Rotate overnight at 4°C.
Washing and extraction
Washing and extraction
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.
Incubate for 5 minutes on ice, repeat pelleting, resuspension and transfer to a fresh tube. Perform this washing step 6 times in total.
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.
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.
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.
Citations
Step 1
Heiman M, Kulicke R, Fenster RJ, Greengard P, Heintz N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).
https://doi.org/10.1038/nprot.2014.085
Step 1
Dougherty JD, Schmidt EF, Nakajima M, Heintz N. Analytical approaches to RNA profiling data for the identification of genes enriched in specific cells.
https://doi.org/10.1093/nar/gkq130
Step 4
Dougherty JD, Schmidt EF, Nakajima M, Heintz N. Analytical approaches to RNA profiling data for the identification of genes enriched in specific cells.
https://doi.org/10.1093/nar/gkq130