DropSeq Droplet Generation Protocol

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DropSeq Droplet Generation Protocol & Tips:

Notes:

  • Keep cells on ice at all times!
  • After breaking the droplets, keep 50 mL bead collection tube on ice as much as possible until reverse transcription.
  • When dealing with beads, be sure to use DNA low-bind Eppendorf tubes. Otherwise the beads tend to stick to the sides of the tube and do not pellet well. This can cause drastic loss of beads if not careful.
  1. Sterile filter 4 mL Droplet Generation oil, load oil into 10 mL syringe, prime tubing with oil
  2. Tape DropSeq chip to side of microscope page directly under the vertical syringe pump
  3. Connect oil channel + outlet channel to DropSeq chip. Ensure all tubing is cut at an angle to facilitate easier insertion. Otherwise, inserting the tubing into the chip is almost impossible.
  4. Make 1M DTT from powder. If 1M DTT is being stored at -20 C, take it out to thaw on ice.
  5. Make 6X SSC if not available. If 6X SSC is in the fridge, place at room temperature.
  6. Prep cells:
    1. Dissociate with Tryp-LE
    2. Neutralize with DMEM-10% FBS
    3. Spin down for 5 min @ 300g
    4. Wash twice with PBS-0.02% BSA (for each wash, spin down for 3 min @ 300g)
    5. Resuspend in plain PBS, place cells on ice. Bring the cells up to the Zhang Lab.
    6. Run cells through 40 um strainer (be sure to wet the strainer with plain PBS first) into a 1.5 mL eppendorf
    7. Count cells using both sides of automated cell counter and average the results. Check the cell size histogram and look at the cells under the microscope to make sure they are single cells (and not clumped in any way)
    8. Make final cell dilution in PBS-0.02% BSA to a final concentration of 100 cells/uL. Make 1.5 mL of this final cell dilution so that it doesn’t run out during the run (you want the lysis buffer solution to be the limiting reagent).
  7. Prep beads:
    1. Spin down bead aliquot (stored in 4 C) for 1 min @ 1000g. All spins involving beads are done for 1 min @1000g.
    2. Resuspend in 1 mL lysis buffer from stock
    3. Add 50 uL 1M DTT, mix by gently pipetting (with 1 mL pipette)
  8. Load cells into syringe, prime tubing, connect to chip
  9. Start magnetic stirring device
  10. Add magnetic stirring disc to syringe, load beads into syringe, connect to chip
    1. When connecting the beads tubing to the chip, make sure the flow never has to travel against gravity. This will always cause a clog. Also make sure there are no kinks in the tubing. These also tend to cause clogging.
  11. Get glass slide + 50 mL collection tube
  12. Start running (cells first, then beads, then oil) and check for leaks immediately
    1. If chip is leaking, switch to a different chip
  13. Wait until outlet stream looks cloudy (droplets are forming), collect outlet on glass slide, view under microscope
    1. Check for droplet quality, bead occupancy
    2. If low droplet quality, check flow rates and try stopping and restarting run
    3. If no beads, check for clogs in bead channel (should see a buildup of white beads in the tubing)
    4. If droplets/beads look good, begin run and set timer
  14. Cool the big centrifuge down to 4 C
  15. Check droplet quality/beads after ~8 min, look for clogs/leaks in the chip
  16. Typical runs will last from 8 to 12 minutes. Make sure one of the channels does not run out of fluid before the others (this will drastically reduce droplet quality)
  17. While waiting for the droplets to collect, begin preparing the reverse transcription mix
  18. After collection is finished, pipette out the oil layer under the droplet layer as per the DropSeq protocol.
  19. Follow standard droplet generation protocol. Generally try to work as fast as possible to limit RNA degradation during this time.