- Before use, thaw the provided Master Plates with Hybridization Master Mixes, vortex the plate in an Eppendorf plate shaker, and centrifuge the plate with a benchtop mini plate-spinning centrifuge or any bucket centrifuge (e.g., 2,000 rpm, 1 min) to collect droplets to the bottom of the wells.
- Remove the seal from the Master plate and transfer 5 µl of Hybridization Master Mix from each well of the Master plate into 1 to 8 new 96-well PCR plate(s) compatible with cell sorter. Importantly, use a new tip for each well of the Master plate to avoid cross-contamination of barcoded reverse GSPs between different wells. A Master plate with wells of unused Hybridization Master Mix can be stored at +4°C for up to 6 months.
- For sorting on the FACS sorter instrument: load the 96-well sorting PCR plate with 5 µl of pre-aliquoted Hybridization Master Mix into the motorized stage holder. Follow the single-cell sorting protocol as per the specific instrument to sort single cells for experimental samples. We recommend including a control well for your plate by sorting 100 cells into 1.2 μL of 1x PBS in one of the wells of this plate. This well can be used as a positive control to ensure cellular RNA is intact after sorting and for troubleshooting purposes.
- Immediately after sorting, remove the plate, seal the plate, and use a benchtop mini plate-spinning centrifuge (or any other centrifuge) to spin the plate for 10-15 secs. This will propel any drops with cells that are on the side walls of the wells down into the buffer. Freeze and store the plate(s) at -80°C prior to use for the scAIR profiling experiment. To reduce sequencing cost, up to 8 plates (e.g., from different experiments) could be run together for the follow-up scAIR-NGS protocol.
Last modified:
7 July 2025
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