In this step, T/B cells sorted or deposited 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, and 96 barcoded mRNA-Rev GSP 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 or cell pools deposited in wells. 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 the 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 a 10-20% excess of reagents, e.g., it is not necessary to increase the volume of reagents to prepare Master Mixes.
- 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 an air incubator), and run the following program to hybridize mRNAs with Reverse GSPs:
Temperature Time 60°C 60 min 25°C ∞
- During Hybridization (Step 1), prepare Diluted SPRIselect beads in the reservoir designed for an 8-channel pipette, mix well the viscous beads and dilution buffer, and keep it at room temperature.
Reagents Volume per plate, µl SPRIselect Beads 300 SPRI bead Dilution Buffer, 1X 300 Total 600
- Add 5 µl (1x volume) of Diluted SPRIselect Beads (prepared in Step 2) into each plate well using an 8-channel pipette, seal the plate, briefly centrifuge the plate, and mix SPRIselect beads and solution in wells using an 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.
- Shake the plate in the Eppendorf plate shaker at 1400 rpm for 10 minutes at room temperature.
- Using an 8-channel pipette, combine the contents of all wells of the 96-well plate together in a reservoir designed for an 8-channel pipette. Transfer the whole volume of the pooled sample into a 1.5 ml test tube. Pipette slowly and try to collect all viscous solutions from wells and reservoirs. Place the 1.5 ml test tube with the pooled sample in a magnetic separation device designed for 1.5 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.
- Add 1 ml of freshly prepared 80% ethanol to 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.
- Repeat 80% ethanol washing in Step 6.
- 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.
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