Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

1. Cloning of transgene, transgenic line production and selection

1. Clone the promoter of choice in the appropriate entry vector. Use a recombination-based cloning method (Table of Materials) and recombine the promoters in pDONRP4-P1r. Clone RPL18 (with affinity tag or fluorescent protein of choice) using recombination-based cloning in pDONRP1-P2⁴⁹.
2. Combine the entry vector containing RPL18 with the promoter-containing entry vector in a two fragment recombination reaction into the appropriate destination vector with FAST-red selection cassette⁵⁰ to facilitate direct selection of transgenic seeds.
3. Verify the recombined vector by sequencing and transform it into suitable, competent agrobacteria. Flower dip Arabidopsis plants and after 3-4 weeks harvest and select T1 seeds⁵¹.
4. Use microscopy to identify well-expressing lines and verify expression patterns according to the reported promoter activity in multiple independent lines. Select lines showing a representative expression pattern with a single T-DNA insertion. This might help to minimize silencing and will be advantageous for genetic crosses.
5. Select T3 offspring that is homozygous for the marker gene.

2. Propagation and sterilization

1. Cell type-specific TRAP isolates RNA from a limited number of target cells per root. To generate the needed starting material, propagate homozygous lines. To this end, use standard growth conditions with a special focus on fungal growth control.
   NOTE: If single insertion lines cannot be obtained, grow batches in large populations over few generations to avoid T-DNA-induced transgenerational silencing.
2. Sterilize large quantities of Arabidopsis seeds with one round of chlorine gas and one round of 70% EtOH.
   1. Spread seeds evenly on 12 cm x 12 cm square Petri dishes (less than 0.3 mL seeds/plate) and stack them into a desiccator or other suitable container. Avoid clump or heap formation as the seeds need to be accessible to the gas. Perform gas sterilization overnight with bleach and HCl volumes as reported⁵²: 100 mL of bleach (13%) with 6 mL of conc. HCl in a 60 L desiccator. Defumigate for at least 1 h before collecting the seeds in a sterile container.
      CAUTION: 37% HCl is highly corrosive and requires careful handling. Chlorine gas is toxic, use a fume hood.
   2. Take 0.1 mL of dry, gas-sterilized seeds per plate and mix them with sterilization solution (70% EtOH, 0,01% Tween) at room temperature. Incubate for 20 min, decant EtOH and wash the seeds 3-4 times with sterile H₂O.
   3. Transfer the soaked seeds into 50 mL tubes and dilute with sterile 0.1% agar to obtain 1 mL of imbibed seed slurry per plate (0.1 mL seed/1 mL slurry).
      NOTE: Due to transgene integration events, plant lines can be susceptible to different sterilization techniques; especially EtOH incubation time was found to be critical. In our hands, the dual sterilization steps were necessary to avoid fungal contamination during the experiments. This is especially important when performing time series as contamination of a single time point hampers the whole experiment. It might well be that dual sterilization is not always needed, depending on local growing conditions.

3. Plating

1. Prepare these steps in advance. Pour ½ MS plates (pH 5.8) with 1% agar in the quantities needed for the experiment (20-30 per sample/time point). Cut 1 mL pipette tips to enlarge the tip diameter to ca. 3-4 mm with a razor blade. Autoclave the tips. Create a template holder for plating three rows of seeds per plate with square Petri dish lids. Prepare a laminar flow hood to provide a sterile work environment and label the plates to be processed.
   NOTE: If many plates are processed at the same time, colored labels can speed up the labeling.
2. Place empty agar plates into the template holder and distribute 1 mL of imbibed seeds evenly onto three rows. Place the processed plates in stacks into the laminar flow until the seeds are dry (i.e., stick to the agar surface). Do not leave the plates longer as the agar will dry out as well.
3. Once the seeds are sufficiently dry, close the lids and seal each plate with micropore tape. Stratify the seeds for two days at 4 °C in the dark and afterwards place them into a growth chamber.

4. Tissue treatment (optional)

NOTE: In this protocol, we outline the exogenous treatment of Arabidopsis roots with the synthetic auxin variant NAA. Depending on the experimental question at hand, this part needs to be adjusted or can be omitted entirely.

1. Prepare strips of tissue paper of 1.5 – 2 cm in height and 10 cm in length. Extended incubation times require the tissue to be autoclaved prior to use.
2. Remove the micropore tape from all plates that have to undergo the hormone treatment. Dilute 1 mL of 10 mM NAA (dissolved in DMSO) in 1 L of liquid, autoclaved ½ MS solution (pH 5.8) and soak the tissue paper in the solution (10 µM NAA).
3. Use tweezers to apply a strip of tissue paper onto each row of roots. Gently use fingers to remove air bubbles. Empty excess liquid from the plate, close the lid and label the plate with the time. For extended incubation times, place the plates back into the growth chamber.

5. Harvesting

1. Retrieve plates for each biological replicate/time point/treatment. Collect liquid nitrogen in a clean Dewar vessel and label tubes (15 or 50 mL) for the different tissue samples. Prepare a Styrofoam holder.
   CAUTION: Become familiar with liquid nitrogen handling procedures (aeration, frostbites, potentially exploding tubes).
2. Open the plate and remove the tissue paper with forceps, being careful not to detach the roots from the agar surface. With a surgical blade, cut once per row along the shoot-root-junction in a single, determined stroke. Clean the blades between samples and exchange frequently to guarantee sharpness.
3. With tweezers, swipe along the roots of each row to collect them in three bundles. Grab the roots and empty them into a 50 mL tube filled with liquid nitrogen to snap freeze.
   NOTE: Do not try to assemble roots into dense structures (like balls) as they are difficult to grind in the next step.
4. Proceed with all plates that constitute one sample (in the order of incubation times) and pour out excess liquid nitrogen. Use the tube lid to prevent the roots from spilling. Then close the lid and collect all tubes in the Dewar vessel. Store the root tissue at -80 °C.

6. Immunopurification

NOTE: This step aims to obtain high-quality TRAP/polysome RNA. Therefore, strictly follow good practice advice for RNA handling. Perform all steps in this section in a sterile bench and clean all equipment and labware with an RNase-removing solution (Table of Materials). Wear gloves and change them immediately when contaminated with sample, ice, or other sources that have not been cleaned. Since this is a very crucial aspect, a section on equipment reuse together with waste disposal advice is included.

1. Buffer preparation
   1. Prepare stock solutions according to Table 1 and autoclave (A) or filter sterilize (₳). Unless otherwise specified, the solvent is RNase-free water.
   2. Dissolve and aliquot dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), cycloheximide (CHX) and chloramphenicol (CAM) in their respective solvents as indicated in Table 1 and store them at -20 °C. All other stocks can remain at room temperature.
   3. Pre-mix the stocks – with ingredients 1-4 for wash buffer (WB) and 1-6 for polysome extraction buffer (PEB) – to avoid time-consuming buffer mixing prior to every extraction. Thus, only add water and the frozen ingredients (7-10) on the day of the extraction. Keep the pre-mixed stocks and RNase-free water at 4 °C.
      NOTE: DTT concentration is 1/5 of the reported concentration from Zanetti et al. 2005, as the nanobody interaction with the GFP is sensitive to high DTT concentrations.

Table 1: Buffer composition and mixing advice. Ingredients with the given stock concentrations mixed in the given amounts yield 50 mL of WB or PEB. Tris: tris-(hydroxymethyl)-aminomethane, EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, PTE: Polyoxyethylene-(10)-tridecyl ether, A: autoclave, ₳: filter-sterilize; *fill up to 50 mL with RNase-free water.

2. Tissue homogenization/grinding
   1. Cool down centrifuge and place homogenizers and centrifuge tubes on ice. Thaw aliquots of DTT, PMSF, CHX and CAM. Mix PEB and WB from the stock solutions in 50 mL tubes according to requirements of the day (# of samples) and cool on ice.
      NOTE: Add PMSF only shortly before use, as the half-life of PMSF in water is only 30 min.
   2. Prepare plenty of liquid nitrogen in a Dewar vessel and retrieve tissue samples from -80 °C storage. Wear cotton gloves underneath the standard lab gloves to prevent burns from cold mortars. Pour liquid nitrogen into mortars and pestles until they are cold enough to allow grinding. It is recommended to devise a system to distinguish mortars (label or keep in a certain order).
   3. Empty tissue sample into a mortar and grind carefully until all material is a white powder. If needed, add liquid nitrogen to keep the tissue frozen or to facilitate better grinding.
   4. Add 5 mL of PEB to the sample and quickly mix with the powder before the buffer freezes. While this sample thaws (mix from time to time) process another sample.
   5. As soon as the mixture can be transferred, empty the slurry into a glass homogenizer and keep on ice. With an additional 2 mL of PEB, rinse the mortar and pestle and add it to the sample in the homogenizer.
      NOTE: Avoid a completely liquid sample as this allows RNA degradation.
   6. Grind the slurry manually until the extract is homogenous. We recommend a minimum of 4 to 5 plunges.
      NOTE: It may require some additional waiting time to allow the slurry to thaw further. Handling of homogenizers requires some diligence. Do not apply brute force and beware of suction forces. If not taken into account, this will lead to spillage, contamination or destruction of the homogenizer.
   7. Pour the crude root extract into a 50 mL centrifuge tube (keep on ice).
      NOTE: Usually several samples can be ground before transfer. Parallel handling of grinding, transferring and homogenizing is required. Try to work quickly but do not rush; stay calm. Always keep homogenized samples on ice.
3. Total RNA sample collection
   1. Transfer 200 µL aliquots of each crude sample to a clean microcentrifuge tube (labeled and cooled on ice beforehand).
   2. Proceed with the RNA extraction as detailed for TRAP samples in points 7.1 and 7.2. Do these steps while samples are clearing in the centrifuge.
   3. Perform a DNase treatment with the resuspended total RNA to eliminate DNA contamination and clean up the reaction using a commercial kit (Table of Materials).
      NOTE: Total RNA extractions usually yield high concentrations and samples need to be diluted considerably. We recommend measuring the concentration after dilution by the sensitive Qubit protocol.
4. Clearing the crude extract
   1. Take the ice bucket with samples from 6.2.7 and centrifuge them for 15 min at 16,000 x g and 4 °C.
      NOTE: To balance out the centrifuge, pair samples accordingly. In case this is not entirely possible, adjust one sample by adding PEB.
   2. Pour the supernatant to a fresh centrifuge tube (cooled on ice beforehand) and repeat centrifugation (15 min at 16,000 x g and 4 °C). This transfer can be quickly performed next to the centrifuge.
   3. While the crude extract is clearing, initiate the washing of GFP-beads for step 6.6.
      NOTE: Keep this ice bucket for rocking on the shaker but do not place back into the sterile bench as it might be contaminated.
5. Bead wash
   1. Aliquot magnetic GFP-beads (#samples x 60 µL, Table of Materials) into a 1.5 mL tube. Place on the magnetic stand. Once the beads have collected, remove the supernatant.
   2. Add 1 mL of cold WB, resuspend the beads and collect them again. Discard the wash buffer and repeat once more with 1 mL of WB.
   3. Ultimately, resuspend the beads in WB to the initial volume used in step 6.5.1.
6. Immunopurification (IP)
   1. Immediately after centrifugation, pour the cleared supernatant into labeled 15 mL tubes and add 60 µL of washed beads per sample.
   2. Place all samples horizontally into the ice bucket and put it on a shaker. Let the mixture incubate for 2 h in order to bind the GFP-labeled polysomes to the beads.
   3. Collect the beads on the magnetic stand for 15 mL tubes (on ice) and add PMSF to the remaining PEB. Discard the supernatant. Pour approximately 5 mL of PEB to the beads and resuspend them by tilting. Shake the samples for 15 min in the same setup as in section 6.6.2.
   4. Repeat the washes with WB to a total of 3 washes (1 x PEB, 2 x WB). Before each buffer exchange, add PMSF.
   5. Collect the beads in 1 mL of WB and transfer them to a 1.5 mL tube. Finally, collect the beads one more time on the magnetic stand and remove all liquid. Close the tube and keep on ice until all samples are processed.
   6. Transport the samples to a fume hood for RNA extraction.
7. Waste disposal and reconditioning of lab supplies.
   1. If performed according to good lab practice (see section 2.2.1), the sterilization procedure yields an aqueous NaCl solution. Leave the chlorine gas, as well as residual HCl and bleach, to defumigate in the fume hood.
   2. PEB and WB disposal: As CHX decomposes at high pH, collect all liquids and bring to pH>9. Dispose of the liquid waste in the halogenated chemical waste. All solids (tissues, serological pipettes, gloves, etc.) should be disposed of as chemical waste.
   3. Collect phenol-containing liquids separately, as well as phenol-contaminated material (tips, tubes and gloves).
   4. Hand-wash mortars, pestles and homogenizers (sponges and brush) with soap and rinse thoroughly. Subsequently, bake the material at >220 °C overnight. Either wrap in tin foil before the treatment or place into a heat-proof, covered container.
   5. Brush clean centrifuge tubes with detergent and then diethylpyrocarbonate (DEPC)-treat in the fume hood. To this end, add liquid DEPC to deionized water (1 mL of DEPC to 1 L of H₂O) and mix via shaking. Place the centrifuge tubes onto an autoclavable tray that catches spilled DEPC-water. Pour the suspension into the tubes and leave for 3 h or overnight. DEPC decomposes in the subsequent autoclaving process.
      CAUTION: DEPC is highly toxic.

7. RNA extraction and QC

1. RNA extraction
   1. Cool down the tabletop centrifuge to 4 °C.
   2. Add 1 mL of acid-guanidinium-phenol-based reagent (Table of Materials) to each sample, invert to resuspend the beads or total RNA slurry and incubate for 5 min on ice. Do not vortex!
   3. Add 200 µL of chloroform and incubate for 3 min on ice. Then thoroughly vortex the samples.
   4. To aid phase separation, centrifuge at max. speed for 10-15 min, 4 °C.
   5. Label 1.5 mL low-retention tubes (Table of Materials) and aliquot 650 µL of isopropanol into each.
   6. Carefully take the upper aqueous phase (ca. 650 µL) and transfer to the prepared tubes with isopropanol. Avoid touching the pink organic phase.
   7. Precipitate RNA overnight at -20 °C.
      NOTE: It is recommended to store the samples in isopropanol at -20 °C or -80 °C and only solubilize in water when needed. Aqueous RNA degrades even at -80 °C when stored for weeks/months.
2. RNA precipitation
   1. Cool down the tabletop centrifuge to 4 °C.
   2. Prepare fresh 80% EtOH with RNase-free water and cool down at -20 °C (5 min at -80 °C help to speed up the process).
   3. Centrifuge the samples at maximum speed (ca. 13,000 x g) for 30 min and discard the supernatant. The pellet will not be visible, so carefully pipette as if it was there. Add 1 mL of cold 80% EtOH and invert the tube one or two times.
   4. Centrifuge again for 30 min at maximum speed and repeat the wash to a total of two washes.
   5. Spin down for 2 min and remove all residual EtOH with a 10 µL tip. Leave the pellet to dry for 3-5 min (not more) at room temperature and resuspend in 20 µL RNase-free water.
   6. Keep the samples on ice and perform quality control as soon as possible. Proceed to store the samples at -80 °C. Avoid freeze-thaw cycles.
3. Quality control using dedicated equipment (Table of Materials) according to the manufacturer's recommendations.

8. Library preparation

1. cDNA synthesis and amplification with the SMARTer v4 Ultra Low Input RNA Kit
   1. Calculate the dilution of each sample to have 1.5 ng of TRAP-RNA or total RNA in a volume of 4.75 µL. Perform all reactions in PCR-tubes and dilute samples with fresh aliquots of RNase-free water.
   2. Perform all steps according to the manufacturer's recommendations with ½ the reaction volumes. Amplify the cDNA with 12-13 PCR cycles.
   3. Clean up the PCR by adding 0.5 µL of 10x lysis buffer and 25 µL of SPRI beads (Table of Materials). If many samples are processed lysis buffer and beads can be pre-mixed. Make sure that the beads are evenly dispersed before pipetting.
   4. Proceed with the protocol in full reaction volumes (17 µL of elution buffer). Do not let the beads dry for more than 3 minutes. Overdried samples can potentially be rescued by prolonged incubation times.
   5. Measure the sample concentrations with the Qubit HS DNA kit.
      NOTE: The SMARTer v4 kit can tolerate down to 200 pg input. We did obtain libraries in cases where Qubit values could not be determined (below 250 pg, detection limit) with a 16 cycle PCR. However, the limited input material might also yield less complex libraries.
2. Fragmentation and adapter ligation PCR with the Nextera XT DNA Library Preparation Kit
   1. Dilute the cDNA with RNase-free water to obtain a concentration of 200 pg/µl and pipette 1.25 µL in a PCR-tube.
   2. Perform all steps according to the manufacturer with ¼ the reaction volumes. Amplify the cDNA with 12 PCR cycles and compatible adapters for the samples that belong to one sequencing pool. With Illumina's Index Kits A and D up to 384 samples can be multiplexed.
   3. For the PCR clean up add 12.5 µL of resuspension buffer and 22.5 µL of SPRI beads (0.9x ratio). Elute the sample with 22 µL of elution buffer.
      NOTE: QC and pooling was performed by the sequencing company (Table of Materials) and thus no bead-based normalization was needed. The enzymatic fragmentation reaction (tagmentation) is very sensitive to material input as every enzyme only cuts once. Therefore, do not exceed the concentration recommendation.