Daniel:Notebook/ComboLock/2016-3-16
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Drop Seq Test (Started 03/15/2016)
After yesterday's results it would appear that we may have polyacrylamide droplets. However, I think the best test is a chemical one, so we'll be following the Macosko protocol for droplet breakage. This protocol uses Perfluoro-1-octanol to break apart droplets. If the droplets did form PA beads, then the octanol shouldn't have an effect. If they didn't the droplets should disappear.
Protocol
- Wash 3X with Mineral Oil (no Span)
- Remove oil phase with a pipette, and add in ~500 uL mineral oil
- Pipette the oil and beads gently to mix
- Repeat 1.1 and 1.2
- Wash 3X with 6X SSC
- Remove supernatant and add 500 uL 6X SSC
- Pipette gently to mix
- Centrifuge for 1 min at 1000xg
- Repeat steps 2.1-2.3
- Add 600 uL of 2% Perfluoro-1-Octanol in 6X SSC (200 uL 20X SSC, 12 uL Perfluoro-1-Octanol, 388 uL ddH2O)
- Shake vigorously by hand for 20 seconds (seriously)
- Centrifuge for 1 min at 1000xg
- Remove supernatant and add an additional 500 uL 6X SSC
- Repeat 3.2 and 3.3 3 times