6.1. Hybridization of mRNA with Reverse C-region Gene-Specific Primers

In this step, mRNA-Rev C GSP hybrids are generated and then purified from non-hybridized primers by nuclease treatment and AMPure magnetic beads (Outline). The protocol is written assuming the reactions will be set off in a 96-well plate. For small numbers of samples, reactions can be done in tubes or strips also. Note: Hybridization, cDNA synthesis, and Forward Primer Extension steps should be set up in a PCR-free room.

1. Prepare a Hybridization Master Mix as described below for all samples and controls (make 5% extra to account for pipetting error) and aliquot 7 µl in the wells of a 96-well plate:
    
   

   Hybridization Master Mix Component	Volume per sample, µl
   Hybridization Buffer, 4X	5
   Reverse TCR-C primer mix, 20X*	1
   Reverse BCR-C primer mix, 20X*	1
   Total	7

*Note: If you need to run only TCR or BCR repertoire analysis, use only TCR-C or BCR-C primer mix and adjust the total volume to 7 µl by water. If you decide to run biological or amplification triplicates, increase the number of tubes, volume, and amount of RNA allocated for each sample respectively. For the DirectCell protocol, add N-Lauroyl Sarcosine to 0.3% (0.7 µl of 20% N-Lauroyl Sarcosine). We recommend running positive control RNA (50 ng), and negative control (water) for troubleshooting and comparing batch effects in different experiments.

2. Adjust the volume of each RNA Sample (e.g., 100 ng of whole blood or 50 ng of PBMC RNA) to 14 µl with water as shown in the table.
    
   

   Component	Volume per sample, µl
   Total RNA (~50-100 ng) of immune cell-rich sample	1 – 14*
   Water, to 14 µl final volume	0 – 13

*Note: For tissue samples with a low content of immune cells, the recommended amount is 200-1,000 ng of total RNA). For AIR profiling directly in immune cell fractions, use 5,000-50,000 cells in 14 µl of 1xPBS buffer.

3. Add 14 µl of RNA Sample to each well with pre-aliquoted 7 µl of Hybridization Master Mix and mix contents by pipetting 3 times. Seal the plate with adhesive film, and spin down to collect droplets. Load the plate in the thermal cycler, and run the following program to hybridize mRNAs with Reverse TCR/BCR-C primers:
    
   

   Temperature	Time
   70°C	5 min
   60°C	60 min
   25°C	∞

4. Add 24 µl (1.2x volume) of Agencourt AMPure® XP Reagent (adjusted to room temperature) to each reaction well, and pipet up and down 5 times to thoroughly mix the bead suspension with the hybridization reaction mix. Check that the whole volume in each well has a uniform brown color.

5. Incubate the mixture for 5 minutes at room temperature. While waiting, prepare the Reverse Transcriptase Buffer Master Mix based on protocol Step 6.2.1. Store on ice before use.

6. Place the plate in the Magnetic Stand for 96-well plates for 1-2 minutes or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.

7. Add 200 μl of freshly prepared 80% ethanol in each reaction well without removal of plate from Magnetic Stand, wait for 2 minutes, carefully remove, and discard the supernatant without disturbing the bead pellets.

8. Repeat 80% ethanol washing in step 9.

9. Briefly centrifuge the plate at low speed and place the plate in the Magnetic Stand. Use a 20-μl pipette to remove the residual ethanol droplets from reaction wells and air-dry the beads at room temperature for approximately 5 minutes.

!Immediately proceed to the next step (cDNA Synthesis)