Protocol for Recycling Beads for Chrono-Seq Device and Preparing Solutions

Best Practices:

1. Remember to put back the reagents back to their storage location at 4°C or -20°C once you are done with using them.
2. Get fresh new microtips/sterile-disposable pipettes after each step, unless mentioned otherwise.
   • Important to avoid cross-contamination.

Equipment Used:

• Our lab uses the refrigerated Sorval ST8R Centrifuge. You can find CAD files to print some of the parts here.
  • We bought the 50ml Inserts and Printed the Rest.
  • You can modify the CAD files to print the 50ml Inserts as well.
  • Use PLA or TPU for the prints.
• CELLTREAT Pipette Controller
  • Replacement Filters
• Eppendorf Pipettes 6-Pack
• Pipette Tips:
  • Genesee Scientific 200/20μl Tips
  • Rainin 1000μl Tips
  • Genesee Scientific 10μl Tips
• Larger Volume Pipettes:
  • 5ml Pipette
  • 10ml Pipette
  • 25ml Pipette
  • 50ml Pipette
• Disposable Sterile Falcon Tubes:
  • 50ml Tubes
  • 15ml Tubes
• Disposable Sterile 250ml GL45 Bottles, Individually Wrapped

Protocol for Preparing Priming Solutions for Chrono-Seq Device.

• Turn on the UV for the Cell Culture Hood and wait 15 minutes.
• Make more 6X SSC Buffer from 20X Stock while you are waiting. Use these 250ml Plastic Bottles.
• Turn on the Cell Culture hood and clean your work area. From this point onwards work aseptically inside the Cell culture hood.
• Inside the Cell Culture hood, make 1X Lysis buffer from the 3.33X Lysis Buffer Stock. You can make 50ml of 1X Lysis buffer by mixing 15 ml of 3.33X Buffer with 35ml of Distilled Water. Please use specially labeled Distilled Water bottles which should only be opened inside the Cell culture hood.
  
• With the Cap closed shake the 1X Lysis buffer vigorously.
• Filter the 1X Lysis buffer using a 40μm Falcon Cell Strainer (Blue Color).
• How many timepoints or injections are you running for your experiment? Change number_of_timepoints_or_injections below to Calculate how many of each Tube you need to prepare:

Optional Note: I recommend adding +1 to number_of_timepoints_or_injections as a backup in case something goes wrong:

number_of_timepoints_or_injections=3 #how many? Include at least one backup in case of Failed Injection.
print("You need to prepare at least",2*number_of_timepoints_or_injections,"50ml Falcon Tubes with 2ml of Lysis buffer each for this experiment.")
print("You also need to prepare at least",number_of_timepoints_or_injections," 50ml Falcon Tubes with 5ml of Distilled Water each for this experiment.")

You need to prepare at least 6 50ml Falcon Tubes with 2ml of Lysis buffer each for this experiment.
You also need to prepare at least 3  50ml Falcon Tubes with 5ml of Distilled Water each for this experiment.

• Transfer 2ml of Lysis buffer each the calculated number of 50ml Falcon tubes.
• Filter a little more than the total Calculated amount of Distilled water with a 40μm Cell Strainer.
• Transfer 5ml of Distilled water each to 50ml Falcon tubes.

Protocol for Recycling Beads.

• Collect all the Tubes with Recovered beads of the same type,batch or timepoint from your previous Experiments.

✶ Important Note: Do not mix different types of beads such as ChronoSeq and Dropseq beads. Also don't mix different timepoints together. It is important you process them for recycling separately.

• Now combine the beads into a single 50ml Falcon Tube inside the Cell Culture Hood. Wash the Tubes with 1X Lysis buffer to get as many beads out as possible.
• Use Compressed Air to Remove any particles from the 50ml Falcon Tubes used for all the steps below.
  • Remove the Particles before moving onto the next step.
  • Execute the Cell Below to Watch a Video of the Process.

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• Now Filter these beads twice with the 70μm Pluriselect Filters (white Color).
  • Use compressed air on the bottom of the filters you will use if you see any particles.
  • You can stack the filters on top of each other. Rotate them 90 degrees with respect to each others axis.
  • From this step onwards its is very important to make sure no particles get into the Lysis buffer with the beads.
  • Add another 5ml of filtered Lysis buffer to the previous container to get any remaining beads out and pass them through the 70μm Filters.
• Spin down the Beads. The Centrifuge should be set to 1000xg for 1.5 minutes with Soft Deceleration Enabled at 21°C.
• Inside the Cell Culture hood, make the total volume approximately 10ml without disturbing the beads.
• Shake the 50ml Tube gently by hand to evenly resuspend the beads.
  • Take a P200 and set it to 20μl. Now using Sterile/Particle-Free 200μl Tips take a sample and transfer it to a Hemocytometer for counting.
  • Make sure the beads are evenly suspended in the Hemocytometer.
• Estimate the Total Volume of Beads in Lysis buffer using a Sterile/Particle-Free 10ml Pipette.
• Calculate the Final Volume of Lysis buffer needed for concentration of 450beads/μl.
• Spin the Beads down. The Centrifuge should be set to 1000xg for 1.5 minutes with Soft Deceleration Enabled at 21°C.
• Remove/Add lysis buffer to get the final desired concentration of 450beads/μl.
  • If you are adding lysis buffer make sure you filter it with a 40μm Cell Strainer before adding.
• Remember to add the following Labels to this Final Tube:
  • Date
  • The Timepoint/Seq-number/Bead-Type
  • The concentration of beads: 450 beads/μl
  • Media in which the beads are suspended: 1X Lysis buffer
  • Final volume of beads in the Tube.

This is a STOPPING STEP. You can store these beads in 1X Lysis Buffer at 4°C.

Protocol for Preparing Bead Tubes for Timepoints/Injections

• You should work inside the Cell Culture hood to avoid introduction of Dust particles or Debris.

✶ Important Note: Do not mix different types of beads such as ChronoSeq and Dropseq beads. Also don't mix different timepoints together. It is important you process them separately.

• For each separate timepoint/bead type (such as Dropseq beads):
  • Label a Sterile/Particle-Free 50ml Falcon Tube with the Bead-Type/Timepoint, the date, the batch and the bead Reservoir Number it will be loaded into for the ChronoSeq Device.
  • Shake the beads each time to make sure the beads have a uniform concentration then, transfer 1ml of Beads each to Separate 50ml Falcon Tubes. Use Sterile/Particle-Free Rainin 1000μl Pipette tips.

  ✶ Important Note:Try be be as close to 1ml of Beads as possible. Less than 0.8ml of Beads can introduce bubbles into the Chrono-Seq device and cause blockages.

  • Put the Beads inside the refrigerator at 4°C before the experiment.
  • Remember to spin down the beads before you load them into the Chrono-Seq device.