Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

1. Culture and metabolic labeling

1. Poke once the center of the cap of a sterile 1.5 mL micro-centrifuge tubes with a 18-gauge needle to allow proper aeration. Inoculate 500 µL of supplemented sterile 7H9 broth with 5 µL of M. smegmatis from a starter culture grown overnight¹².
   NOTE: The recipe for one liter of 7H9 broth is: 4.7 g Commercial 7H9 powder, 2 mL Glycerol, 1 mL Tween 80, 0.85 g NaCl, 5 g Albumin, 2 g Dextrose, H₂O (to bring total volume to 1 L).
   1. (CAUTION) Following standard radioprotection practices, spike the culture media with 20 µCi/mL of [³²P] Na₂HPO₄. Grow bacteria at 37 °C and 1,200 rpm agitation, in a shaker incubator placed behind a 9-mm thick acrylic shield.
2. At midlog phase (O.D.600 nm ~0.5), transfer the entire culture into a 2-mL screw-cap tube and pellet radioactive bacteria at room temperature by centrifuging for 2 min at 10,000 x g. Collect supernatant containing unincorporated radioactive orthophosphate in appropriate waste container.

2. Preparation of total RNAs

NOTE: Total RNAs are extracted from bacterial pellets using a commercial RNA extraction reagent (containing guanidinium thiocyanate, phenol and chloroform) following the manufacturer's protocol.

1. Add 1 mL of commercial RNA extraction reagent and approximately 200 µL of glass beads onto the pellet. Cap the tube securely and disrupt the bacterial in a homogenizer for 2 min under maximum agitation, (setting number five on chosen instrument (see the Table of Materials)).
2. Centrifuge sample at 12,000 x g for 10 min at 4 °C. Transfer the supernatant to a new 2-mL tube and discard the beads appropriately. Add 0.2 mL of chloroform and shake tube vigorously by hand for 15 s. Centrifuge at 12,000 x g for 15 min at 4 °C.
3. Collect the aqueous phase of the sample into a new tube by angling the tube at 45°. Avoid drawing any of the interphase or organic layer. Add 2 µL of colored precipitant and 0.5 mL of 100% isopropanol. Shake the tube vigorously by hand for 5 s. Centrifuge at 12,000 x g for 10 min at 4°C.
4. Remove the supernatant from the tube and discard appropriately as the liquid fraction may contain traces of radioactivity. Air dry pellet for 5 min then resuspend in 200 µL of 2X saline-sodium citrate (SSC), 0.1% (w/v) sodium dodecyl sulfate (SDS) final.
   NOTE: The composition of stock 20 X SSC is 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0.

3. Preparation of 96 well printer plates

NOTE: tRNA microarrays are manually printed with forty two 70-mer DNA oligonucleotides complementary to the 3' end of each M. smegmatis tRNA (terminal CCA excluded) using an eight-pin arrayer. The sequences of the DNA oligonucleotide probes as well as the probe layout in the well-plate and the array are provided (Supplementary File 1- sheet 1 to 3 respectively).

1. Design DNA probes by first retrieving tRNA sequences or tRNA encoding genes from tRNA databases such as the Genomic tRNA Database¹³. Trim conserved 3' end CCA if encoded. Reverse complement the resulting sequence. Order probe based on the first 70 nucleotides.
2. Resuspend dry DNA probes in water. Adjust probes concentration to 100 µM. In a 96 well-plate, prepare 120 µL of 50 µM oligos in 3X SSC, 0.01% (w/v) SDS final (dilute 60 µL of stock probes in individual well with 60 µL of 6X SSC , 0.01% (w/v) SDS.
3. Cover plate with tin foil to prevent evaporation or contamination, freeze immediately and keep at -20 °C until needed.

4. Array printing

1. Thaw the 96-well plate at room temperature (takes approximately 20 min).
2. Label amine-coated glass slides with a diamond pen. Place slides into indexing unit. Follow the grid placements and do as follows.
   1. Carefully dip the replicator pins in the wells.
   2. Gently print the array on the glass slides, using minimal pressure (you will feel a small resistance). Continue printing without cleaning the arrayer until finished with block A.
3. When ready to move on to the next block, follow the washing procedure described below.
   1. Dip replicator in the 5% bleach and gently shake.
   2. Press replicator into absorbent paper.
   3. Dip replicator in the distilled water, gently shake, and press into paper. Repeat this step once.
   4. Dip replicator in isopropanol, gently shake, and press into the paper.
   5. Dry the replicator on the fan for about 20 s before moving on to the next block of the 96-well plate. Continue to desired number of prints.
4. Allow the slides to dry then crosslink the oligos to the slide using a 254-nm UV crosslinker.
   1. Set the energy level to 999,990 µJ/cm².
   2. Put the slides face up on a clean surface in the crosslinker. Press "start".
5. Block the arrays overnight in 450 mL of blocking solution on a magnetic stirrer, at room temperature and under slow agitation. Collect and reuse blocking solution up to four times.
   NOTE: The recipe for the blocking solution is: 125 mM phosphate buffer (NaH₂PO₄/Na₂HPO₄-) pH 7.2, 2.25% SDS (w/v), 0.5 mM EDTA, 0.5X SSC, 1% BSA (w/v).
6. Wash the slides 2 times for 5 min at room temperature in 500 mL of distilled water. Dry the slides by centrifuging for 10 s at room temperature in a microarray centrifuge. Store arrays in a dry and dark place for up to 6 months.

5. Array hybridization

1. Prior to hybridization rinse slides in boiling water and dry by centrifugation (as in 4.6).
2. Place array inside a hybridization cassette and load radiolabeled RNA sample.
3. Run the automated hybridization-washing station for maximum reproducibility.
   1. Program steps as following. Condition gaskets for 2 min at 75 °C. Introduce probe at 60 °C.
      Denature probes and samples 5 min at 90 °C. Hybridize samples for 3 h at 60 °C.
      1. Wash slides at 50 °C twice with 2X SSC, 0.1% (w/v) SDS, flow 10 s and hold 20 s. Wash slides at 42 °C twice with 0.1X SSC, 0.1% SDS, flow 10 s and hold 20 s. Wash slides at 42 °C twice with 0.1 X SSC, flow 10 s and hold 20 s.
4. Dry slides by centrifugation (as in 4.6).

6. Array quantification

1. Wrap slides using a thin plastic film. Check slides for radioactive signal using a Geiger counter on its most sensitive setting. Expose slides onto a storage phosphor screen in an exposure cassette at room temperature for 10 to 90 h depending on signal strength.
   NOTE: As sensitivity of Geiger counters vary from one instrument to another, exposure times must be optimized empirically.
2. Scan slide at 50 µm resolution using a phosphorimager. Quantify and background-subtract radioactivity intensities at each probe spot using the free Image J software upgraded with a microarray profiler.
3. Organize and process data using an electronic spreadsheet. Generate heat maps using the conditional formatting tool. Represent for example each probe signal as a fraction (expressed in ‰) of the total probe signal.