6.1. Hybridization with Rev-GSP Primers

In this step, T/B cells sorted in the 96-well PCR plate(s) are incubated in a hybridization buffer containing barcoded Rev GSPs at high temperatures. In this hybridization step, mRNA-Rev GSP hybrids are generated, purified from nonhybridized primers by nuclease treatment, 96 barcoded mRNA-RevGSP hybrids are combined and purified with SPRIselect magnetic beads. If you run more than one 96-well plate with sorted single T/ B cells, each plate gives one pooled reaction tube, i.e., don’t combine pooled samples from different plates. The protocol below specifies the volume of reagents for processing one 96-well plate with sorted single T/B cells. The same protocol could be used for processing the plate with a Positive Control RNA sample or a Positive Control RNA sample spiked with TCR/BCR synthetic controls (in each well). The scAIR kit provides enough reagents to process up to 8 Pooled samples (each from a 96-well plate). In current and all follow-up steps proportionally increase the volume of reagents to assemble Master mixes if you are using more than one plate.

Please, note that the proposed volumes of Master Mixes for all steps already include 10-20% excess of reagents, e.g., it is not necessary to increase the volume of reagents to prepare Master Mixes.

*Note: Hybridization, cDNA synthesis, and Forward Primer Extension steps should be set up in a pre-PCR room.

1. Thaw the plate with sorted cells (stored at -80°C, Section 5.1) and briefly centrifuge the plate to collect droplets. Load the plate in the thermocycler (or in air incubator), and run the following program to hybridize mRNAs with Reverse GSPs:
    
   

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

2. During the hybridization step, prepare the Primer Removal Master Mix:
    
   

   Primer Removal Master Mix Component	Volume per plate, µl
   Primer Removal Buffer, 10X	54
   Water	432
   Primer Removal Enzyme	54
   Total	540

3. Aliquot 66 µl of prepared Master Mix in each tube of the 8-tube strip, and store the 8-tube strip at +4°C:

4. After completion of the Hybridization step, using an 8-channel pipet add 5 µl of Primer Removal Master Mix (prepared in Step 2) on the side wall of each plate well. Seal the plate, mix the reagents in the plate using an Eppendorf plate shaker, and briefly spin the plate to collect the contents at the bottom of the wells. Incubate the plate in a thermocycler (or in an air incubator) at 37°C for 20 min.

5. During the Primer Removal step (Step 3), prepare Diluted SPRIselect beads in the reservoir designed for an 8-channel pipet, mix well viscous beads and dilution buffer, and keep it at room temperature.
    
   

   Reagents	Volume per plate, µl
   SPRIselect Beads	300
   SPRI bead Dilution Buffer, 1X	900
   Total	1200

6. Add 10 µl (1x volume) of Diluted SPRIselect Beads (prepared in Step 4) into each plate well using an 8-channel pipet, seal the plate, briefly centrifuge the plate, and mix SPRIselect beads and solution in wells using Eppendorf plate shaker. Importantly, you need to optimize the speed of the plate shaker (approximately 1,400-1,800 rpm) to completely mix viscous SPRIselect beads with a solution in each well. Check visually that each well contains a homogeneous brown color solution in the whole volume.

7. Shake the plate in the Eppendorf plate shaker at 1400 rpm for 10 minutes at room temperature.

8. Using an 8-channel pipet combine the content of all wells of the 96-well plate together in a reservoir designed for an 8-channel pipet. Transfer the whole volume of the pooled sample into a 2 ml test tube. Pipet slowly and try to collect all viscous solutions from wells and reservoirs. Place the 2 ml test tube with the pooled sample in a magnetic separation device designed for 1.5-2 ml test tubes for approximately 5 min until the liquid appears completely clear. Carefully remove and discard the clear supernatant from the test tube without disturbing the magnetic bead pellet.

*

• Note: If you run several 96-well plates, each 96-well plate content needs to be pooled separately as each plate has the same set of 96 well-specific indexes in Rev GSPs. .
  

9. Add 2 ml of freshly prepared 80% ethanol in the pooled sample test tube without removing the test tube from the Magnetic Stand, wait for 2 minutes, carefully remove, and discard the supernatant without disturbing the bead pellet.

10. Repeat 80% ethanol washing in Step 9.

11. Briefly centrifuge the pooled sample test tube at low speed and place the test tube in the Magnetic Stand. Use a 200-μl pipette to remove the residual ethanol droplets from the bottom of the test tube and air-dry the beads at room temperature for approximately 5 minutes until the disappearance of ethanol droplets on the test tube surface and the magnetic bead pellet appears dry and no longer shines.

• Note: During the process of drying the magnetic beads, prepare the Reverse Transcriptase Master Mix as described in the next section.

!Immediately proceed to the next step (cDNA Synthesis)