- 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. Depending on your experimental design, you can also sort more than one cell into some wells. For example, you could consider sorting up to 500 bulk control cells in one well as a strategy to identify the most abundant clonotypes and/or to use as a positive control to troubleshoot the protocol.
- 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:
15 May 2025
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