Blue:RNA-Seq Experiments:02272014

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Human Nuclei C1 Experiment: 02-27-2014[edit]

Purpose[edit]

  • To test conditions for preventing Nuclei clumping during C1 loading


Tests[edit]

  • Sort 50K nuclei per tube in 1% BSA buffer
  • Centrifuge 910g at 4C for 10 min
  • Resuspend in 1% BSA sort buffer containing:
  • No supplements
  • 1mM MgCl2
  • 25% Glycerol
  • 10% PEG6000
  • 1mM MgCl2/25% Glycerol
  • 1mM MgCl2/10% DMSO
  • Count using Biorad T20:
  • No suppl - 9.75x10e5
  • 1mM MgCl2 - 9.03x10e5
  • 25% Glycerol - 9.87x10e5
  • 10% PEG - 9.78x10e5
  • 1mM MgCl2/25% Glycerol - 9.2x10e5
  • Add 7ul to 3ul C1 Suspension RGT
  • Incubate 10 min at RT
  • Centrifuge @910G for 10 min
  • Gently resuspend and add into BioRad slide for counting/sizing
  • No suppl - 5.93x10e5
  • 1mM MgCl2 - 6.79x10e5
  • 25% Glycerol - 7.97x10e5
  • 10% PEG - 0x10e5
  • 1mM MgCl2/25% Glycerol - 6.93x10e5

Results: PEG caused a lot of precipitation and the nuclei looked damaged - a lot of multiples were observed. BSA sort buffer alone did not show any significant clustering with only a few duplicates observed. MgCl2 and glycerol did not seem to improve anything, potentially more duplicates with MgCl2. Therefore, the issue with clumping may be batch dependent and can be checked in the future by observing the results of the count with the BioRad T20: (1) ensuring the histogram doesn't extend past 10um in nuclei size (since previous failed sorts showed the peak extending up to 16um - likely clumps)and our histograms here only extended to 10um; (2) Checking the counted nuclei under the microscope to ensure no clumps are observed

  • Loaded 800 nuclei from BSA condition alone onto C1 and added 10% DMSO to the rest for storage at -80C
  • Stored 25% glycerol condition at -20C overnight