Materials and Reagents

Microcentrifuge tube
Streptavidin MyOne T1 Dynabeads (Thermo Fischer, catalog number: 65602). Store at 4°C
Purified Recombinant Biotinylated Protein L (Pierce, catalog number: 29997). Store at 4°C
Bovine Serum Albumin (BSA) (IgG-Free, Protease-Free) (Jackson ImmunoResearch, catalog number: 001-000-162). Store at 4°C
Cycloheximide (CHX) (Sigma, catalog number: C7698). Store at -20°C
1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) (Avanti Polar Lipids, catalog number: 850306P-200mg). Store at -20°C
DL-Dithiothreitol (DTT) (Sigma, catalog number: D9779). Store at -20°C
Pierce Protease Inhibitor Tablets, EDTA-Free (Pierce, catalog number: A32955). Store at 4°C
Recombinant RNasin Ribonuclease Inhibitor (Promega, catalog number N2518). Store at -20°C
SUPERase-In (Invitrogen, catalog number: AM2694). Store at -20°C
1 M HEPES solution pH 7.0–7.6, sterile filtered (Sigma, catalog number: H0887). Store at 4 °C.
10× HBSS without calcium chloride, magnesium chloride or magnesium sulfate (Gibco, catalog number: 14185-052). Store at room temperature
UltraPure Distilled Water, DNase, RNase Free (Invitrogen, catalog number: 10977-015). Store at room temperature
Dulbecco’s Phosphate Buffered Saline (PBS) (Gibco, catalog number: 14190-144). Store at 4°C
2 M KCl RNase free (Invitrogen, catalog number: AM9640G). Store at room temperature
1 M NaHCO₃ (Sigma, catalog number: S5761-500G), sterile filtered. Store solution at 4°C
1 M MgCl₂ RNase free (Invitrogen, catalog number: AM9530G). Store at room temperature.
GFP antibodies (Memorial Sloan-Kettering Monoclonal Antibody Facility; clone names: Htz-GFP-19F7 and Htz-GFP-19C8). Store at -80°C
Glucose (Sigma, catalog number: G7021). Store at room temperature
Sodium Bicarbonate (Sigma, catalog number: S6297). Store at room temperature
β-mercaptoethanol (β-ME) (Sigma, catalog number: 63689). Store at room temperature in flammable hood
NP40, IGEPAL CA-630 (Sigma, catalog number: 56741). Store at room temperature
Ethanol (American Bioanalytical, catalog number: AB00138-01000). Store at room temperature in the flammable hood.
ChromoTek TRAP kit (ChromoTek, catalog number: gtmak-20). Store at 4°C
Qiagen RNeasy Plus Mini Kit (Qiagen, catalog number: 74134)
TRAP transgenic mice, (Gt(ROSA)26Sor^{tm1(CAG-EGFP/Rpl10a,-birA)Wtp}, Jax^#022367)
Dissection Buffer (1×, 150 mL) (see Recipes)
Homogenization buffer (see Recipes)
Low Salt buffer (see Recipes)
300 mM DHPC (see Recipes)
High Salt buffer (see Recipes)

Equipment

DynaMag-2 Magnet (Life Technologies, catalog number: 12321D)
Teflon-glass homogenizer with serrated PTFE pestle (DWK Life Sciences, catalog number: 8855100020)
Refrigerated Centrifuge (Eppendorf, Centrifuge 5430R)
End over end microcentrifuge tube rotator
Microcentrifuge (Eppendorf, Centrifuge 5415D)
Nanodrop One (Thermo Scientific, catalog number: ND-ONE-W)
RNase free 1.7mL microcentrifuge tubes (Genesee Scientific, catalog number: 24-282)

Software

R v 4.02, https://cran.r-project.org

Procedure

Note: The following protocol was optimized to characterize the mRNA translation within cerebellar Purkinje cells. For each purification, 30 µL of Streptavidin MyOne T1 Dynabeads, 12 µL of biotinylated protein L, and 50 µg of each GFP antibody (100 µg total antibody) were utilized to perform the TRAP experiments. Please include a 0.5–1-fold excess of the reagents, to account for pipetting errors of the matrix components.

Depending on the abundance of your target cell type and the amount of available sample (i.e., volume of cortex versus hippocampus), you may need to adjust the volumes of bead/protein L matrix, while keeping the ratio (2.5:1 beads/protein L) the same to optimize the capture of the translated RNA from the GFP-tagged ribosomes (Heiman et al., 2014). Preliminary pilot experiments are recommended, to confirm that you are using the optimal volume of the bead/protein L matrix to characterize your target cell type, while allowing for differences in translational state and tissue background RNA binding levels. Before performing RNAseq on the TRAP-isolated samples, confirm targeted enrichment by completing qPCR of specific genes known to be enriched in your desired sample.

Bead/Matrix Preparation (Takes approximately 3 h for preparation)
Note: The matrix (beads/protein L and antibodies) should be prepared as a common stock and then aliquoted into the individual tubes in section C, just prior to the addition of the isolated S20 samples.
We typically prepared the matrix fresh on the day of the experiment, however, if needed, it can be prepared in advance and stored for up to 1–2 weeks at 4°C in the presence of 0.02% sodium azide. If using pre-prepared matrix, wash the matrix stock three times with low salt buffer (see Recipe 3) prior to aliquoting for use in section C (Heiman et al., 2014).
1. Re-suspend Streptavidin MyOne T1 Dynabeads by gentle hand mixing/pipetting (do not vortex). Wash Streptavidin MyOne T1 Dynabeads (30 µL per sample) twice with 1× phosphate buffer solution (PBS) at room temperature (1 mL for all washes, if using a 1.5 mL microcentrifuge tube). Collect beads on magnet (approximately 30–60 s until the supernatant runs clear), and discard the supernatant.
2. Re-suspend beads in 1× PBS using the appropriate volume (original bead volume minus the volume of biotinylated protein L to be added, still accounting for fold-excess integer to allow for pipetting error).
3. Add Purified Recombinant Biotinylated Protein L (12 µL per sample) to suspended Streptavidin MyOne T1 Dynabeads beads. Rotate end over end at room temperature for 35–45 min.
4. Collect the beads coated with protein L on the magnet. Wash the protein L-coated beads five times with 1× PBS containing 3% (weight/volume) BSA. Collect on magnet with each wash, and discard supernatant.
5. Wash the protein L-coated beads three times with 500 µL of low salt buffer (Recipe 3). Collect on magnet with each wash, and discard supernatant (see Figure 1).
6. Incubate protein L-coated beads with anti-GFP antibodies (50 µg each of HtzFFP-19F7, and HtzGFP-19C8) diluted in 100 µL of low salt buffer end over end at room temperature for at least 1 h.
7. After incubating with anti-GFP antibody mixture, wash the beads three times in low salt buffer. Collect on the magnet with each wash, and discard the supernatant.
8. Re-suspend the antibody-coated beads with protein L in an aliquot of low salt buffer, containing 300 mM DHPC (Recipe 4), to maintain the desired ratio of beads/protein L/GFP antibody just before adding the S20 lysate (Procedure C).
Option 2: Use the ChromoTek TRAP Kit, which provides a pre-prepared GFP-Trap Agarose affinity resin for immunoprecipitation of GFP-fusion proteins. It also contains the wash and elution buffers, as well as a lysis buffer that can be used on mammalian cells, but not for tissue.
1. Resuspend provided beads by gently pipetting up and down, and/or inverting the tube. Do not vortex the beads.
2. Transfer 50 µL of the slurry per sample into a 1.5-mL microcentrifuge tube. Add an extra sample (i.e., 50 µL) to the total, to allot for pipetting error.
3. Add 500 µL of ice-cold dilution buffer (provided in the ChromoTek TRAP kit) to the beads.
4. Separate the beads with the magnet, until the supernatant is clear. Discard the supernatant.
  Note: If you obtain the non-magnetic agarose beads, you can follow the same preparation, but rather than separating the beads out on the magnet, separate the beads by centrifugation at 2,000 × g and 4°C for 3 min.
Figure 1. Illustration of how to wash magnetic TRAP beads. Place Eppendorf tube on magnet (A). Allow supernatant to clear, and then pipet off the supernatant, without dislodging the beads form magnet (B).
Dissection of brain tissue
1. Obtain and weigh transgenic animals.
2. Euthanize animals with CO₂, and decapitate. Remove and weigh brain (if desired) prior to dissection.
3. Rinse brain with dissection buffer (Recipe 1), and then rapidly dissect out cerebellum (or area of interest, i.e., cortex, hippocampus) in ice-cold dissection buffer.
4. Place approximately 25–50 mg of dissected tissue in 1 mL of ice-cold homogenization buffer (Recipe 2). Flash freeze the rest of the brain for controls/additional experiments in the future.
  Notes:
  1. Depending on the abundance of your desired cell type, you may have to decrease or increase the amount of tissue that you place in the homogenization buffer.
  2. While you can flash freeze the dissected area of interest of the brain with liquid nitrogen, and store at -80°C prior to homogenization and completion of the protocol, your yield of purified mRNA may not be as high. Thus, for populations of cells with lower abundance, we recommend performing the experiment on fresh lysates. If you do use frozen tissue, do not thaw the tissue, but rather place it directly into the ice-cold homogenization buffer, and proceed with the tissue homogenization (the tissue will thaw during the homogenization).
5. If desired, save a piece of the animal’s tail or ear for re-genotyping, to confirm the experimental genotype.
Homogenization/Preparation of lysates
1. Homogenize the cerebellum (~25–50 mg of tissue) in 1 mL of ice-cold homogenization buffer, using 12 motor-driven strokes of Teflon-glass homogenizer.
  Note: Keep the pestle in the liquid to avoid excessive aeration/formation of bubbles, which can result in protein denaturation.
2. Transfer the homogenized lysate to a pre-chilled microcentrifuge tube.
3. Centrifuge at 2,000 × g and 4°C for 10 min to remove nuclei.
4. Transfer supernatant to new pre-chilled microcentrifuge tube. Add 1/9 volume of 10% NP-40 (1% final concentration), and invert five times. Then add 1/9 volume of 300 mM DHPC (final concentration 30 mM DHPC), invert 5 times, and incubate on ice for 15 min.
5. Centrifuge at 20,000 × g and 4°C for 15 min, to remove mitochondria. The resulting supernatant (S20) contains the polysome fraction. Place the S20 fraction in a new pre-chilled microcentrifuge tube.
6. Save a small aliquot (~50–60 µL) of S20 as an input control. Add low salt buffer to the S20 input sample (to make the final volume up to 250 µL), and incubate at 4°C overnight, along with the immunoprecipitation samples (as in section D). Following incubation overnight, add 750 µL of Trizol, and place at -80°C, or extract RNA via the Qiagen RNAeasy kit described in Procedure D, Option 2.
Immunoprecipitation/RNA preparation
1. Add 200 µL of GFP-conjugated beads to the remaining S20 lysate for each sample.
  Note: Remember to gently re-suspend the bead/protein L/GFT antibody matrix, by gentle pipetting immediately prior to aliquoting and adding the S20 lysates.
2. Incubate end-over-end in the cold room (4°C) overnight.
3. Collect the beads on the magnet. Save a small aliquot of the supernatant, which represents the unbound fraction. Wash beads four times in 1 mL of high salt buffer (Recipe 5). Be careful not to introduce bubbles when pipetting during the washes. Collect on the magnet, and discard the supernatant.
4. Re-suspend beads in 250 µL of low salt buffer, and 750 µL of Trizol. Place at -80°C. Prepare RNA as per Trizol protocol when you are ready to proceed.
  Note: You can also use the Qiagen RNAeasy kit as described in Option 2, rather than Trizol.
Option: ChromoTek TRAP Kit
1. Add the entire S20 lysate to 50 µL of equilibrated ChromoTek TRAP beads (pre-equilibrated in Section A).
2. Rotate end-over-end in the cold room (4°C) for 1 h.
3. Separate the beads with a magnet until the supernatant is clear (or centrifuge at 2,000 × g and 4°C for 3 min, if using agarose beads). Save 50 µL of supernatant for further analysis of non-bound/flow-through fraction.
4. Discard the rest of the supernatant.
5. Wash beads five times with high salt buffer, and separate beads either on magnet, or via centrifugation (2,000 × g at 4°C for 3 min), if using agarose beads.
  Note: You can use the provided wash buffer that comes with the kit, but we tended to have higher yields of RNA using the high salt buffer.
6. After final wash, remove supernatant, and add 350 µL of RLT buffer (Qiagen RNAeasy kit) containing β-mercaptoethanol (β-ME) (10 µL of β-ME for 1 mL of RLT). Incubate at room temperature for 10 min.
7. Depending on your formulation of ChromoTek TRAP beads, either separate beads using the magnet, or centrifuge samples at 2,000 × g and 4°C for 3 min. Transfer the supernatant to a new tube.
8. Add 1× volume of 70% ethanol to the tubes, and mix by pipetting.
9. Load 700 µL of the mixture onto the provided Qiagen column. Centrifuge at 8,000 × g at room temperature for 15 s. Discard the flow-through.
10. Add 700 µL of RW1 (Qiagen kit) to the column. Centrifuge at 8,000 × g at room temperature for 15 s. Discard the flow-through.
11. Add 500 µL of RPE (Qiagen kit) to the column. Centrifuge at 8,000 × g at room temperature for 15 s. Discard the flow-through.
12. Add a second 500 µL of RPE (Qiagen kit) to the column. Centrifuge at 8,000 × g at room temperature for 2 min. Discard the flow-through.
13. Place the RNeasy spin column in a new 2-mL collection tube, and centrifuge at maximum speed for 1 min, to dry the membrane.
14. Place column in a 1.5-mL microcentrifuge tube, and add 30 µL of RNA-free water directly to the column membrane, to elute the RNA. Centrifuge at 8,000 × g at room temperature for 1 min.
15. Take the eluant, and place it back on the column, and spin at 12,000 × g for one minute.
16. Determine the concentration of the RNA by Nano-drop.
Sequencing
1. cDNA libraries must be constructed prior to sequencing, and there are many commercially available kits for this procedure, as well as many institutions which have core facilities that will perform this service. The main considerations when choosing the kit are mRNA selection method, and starting RNA concentration. Library barcodes can be added during preparation to allow for pooling and multiplexing of samples.
2. Sequence the pooled libraries on an Illumina platform.

Data analysis

Quality control of sequencing run
1. Make sure that sequencing run parameters are tolerable (>80% clusters passing filter, >90% Q30). Deviations from these parameters suggest an issue with the sequencing run itself, and can be related to inaccurate quantification of the cDNA library.
2. Run FastQC on each sequencing file.
Aligning and quantifying reads
1. Obtain the appropriate version of the genome
  wget https://ftp.ensembl.org/pub/release-105/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.primary_assembly.fa.gz
  gunzip Mus_musculus.GRCm39.dna.primary_assembly.fa.gz
  wget https://ftp.ensembl.org/pub/release-105/gtf/mus_musculus/Mus_musculus.GRCm39.105.gtf.gz
  gunzip Mus_musculus.GRCm39.105.gtf.gz
2. Create a STAR index for mapping
  STAR --runThreadN 8 --runMode genomeGenerate --genomeDir ./GenomeDir --genomeFastaFiles Mus_musculus.GRCm39.dna.primary_assembly.fa
  --sjdbGTFfile Mus_musculus.GRCm39.105.gtf
3. Align sequencing files to genome using STAR
  STAR --runThreadN 8 --genomeDir ./GenomeDir --readFilesIn Sequencing_1.fastq.gz gz --readFilesCommand gunzip -c --sjdbGTFfile Mus_musculus.GRCm39.105.gtf --outFileNamePrefix Sequencing_1 --outSAMtype BAM SortedByCoordinate
4. Assemble transcriptome using Cufflinks
  cufflinks -p 8 -g Mus_musculus.GRCm39.105.gtf --library-type fr-firststrand -o ./Sequencing_1 Sequencing_1Aligned.sortedByCoord.out.bam
5. Merge transcriptomes
  cuffmerge -p 8 -g Mus_musculus.GRCm39.105.gtf assembly_list.txt
  *assembly_list.txt contains a list of the transcripts.gtf files created by cufflinks
6. Quantify transcripts
  cuffquant -p 8 --library-type fr-firststrand -o ./Sequencing_1 ./merged_asm/merged.gtf Sequencing_1Aligned.sortedByCoord.out.bam
  *Can also use a custom transcript database. For example, we generated the transcriptome using RNA sequencing data from sorted Purkinje Cells, and then we used that .gtf file to annotate transcripts identified in the TRAP data. This greatly facilitated comparison of transcriptome and TRAP data.
7. Create a matrix of gene expression
  cuffnorm -p 8 --library-type fr-firststrand -o ./output -L Sequencing_1,Sequencing_2,… Sequencing_1/abundances.cxb Sequencing_2/abundances.cxb …
8. This has created a gene expression matrix across all samples at the gene and transcript level. From here, several analyses are possible, and should be informed by the biological questions posed in the study. One analysis of interest might be to find transcripts that show altered ribosomal binding between conditions. For this analysis, we used LIMMA to identify transcripts showing changes between mutant and control Purkinje Cells. If transcriptome data is available for the sample, then it may be of interest to compare changes at the ribosomal binding level to those at the transcriptome level. In order to do this, we calculated the translation efficiency for each condition, and used the change in translation efficiency to compare between conditions.

Notes

All solutions must be EDTA-free, as EDTA chelates magnesium and will lead to the dissociation of polysomes.

Recipes

Dissection Buffer (1×, 150 mL)
10× HBSS, 15 mL Final 1× HBSS
1 M HEPES, 375 µL Final 2.5 mM HEPES KOH pH 7.4
1 M Glucose, 5.25 mL Final 35 mM Glucose
1 M NaHCO₃, 600 µL. Final 4 mM NaHCO₃
RNase Free water, 128.775mL
Add CHX 100 µg/mL (freshly made) to desired volume
Note: CHX stock is dissolved in methanol for lysates, and DMSO for cultured cells.
Homogenization buffer
10 mM HEPES pH 7.4, 5 mM MgCl₂, 150 mM KCl, 0.5 mM DTT, 100 µg/mL CHX, 10 µL/mL RNasin, 10 µL/mL SUPERase-In, 1× complete EDTA-free protease inhibitors
Low Salt buffer
0.15 M KCl buffer (10 mM HEPES pH 7.4, 5 mM MgCl₂, 150 mM KCl, 1% NP40)
300 mM DHPC
Adding 1.38 mL 10 mM HEPES pH 7.4 to 200 mg DHPC powder
High Salt buffer
0.35 M KCl buffer (20mM HEPES pH 7.4, 5mM MgCl₂, 350 mM KCl, 1% NP40, 0.5M DTT, 10 µL/mL RNAsin, 10 µL/mL SUPERase-In, and 100 µg/mL CHX)

Acknowledgments

We would like to acknowledge the original paper from which this protocol was adapted from Heiman et al. (2014) “Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP)” (Heiman et al., 2014), as well as our work from which this protocol is derived from Dalal et al. (2021; DOI: 10.7554/eLife.67399), “Loss of Tsc1 in cerebellar Purkinje cells induces transcriptional and translation changes in FMRP target transcripts.” (Dalal et al., 2021). The Sahin lab is supported by grants from the NINDS R01NS113591 and from the U.S. Army Medical Research Tuberous Sclerosis Complex Research Program (W81XWH-15-1-0189) and the Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center (BCH IDDRC, P50HD105351). CLS is funded by the CH/BIDMC/Harvard Medical School Neurology Resident Research Education Program NIH (R25NS070682). KDW is funded by a NIH K08 (K08NS112598).

Competing interests

CLS and KDW do not have any financial or non-financial competing interests. Mustafa Sahin reports grant support from Novartis, Roche, Biogen, Astellas, Aeovian, Bridgebio, Aucta and Quadrant Bio- sciences. He has served on Scientific Advisory Boards for Novartis, Roche, Celgene, Regenxbio, Alkermes and Takeda.

Ethics

All animal procedures were carried out in accordance with the Guide for the Humane Use and Care of Laboratory Animals. All the procedures in this protocol were approved by the Animal Care and Use Committee of Boston Children’s Hospital (protocol number 18-05-3677R).

References

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