Blue:RNA-Seq Experiments:07312013

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Purpose[edit]

  • Fluidigm C1 on mouse NPCs (E12.5) (Yun Yung prepared - J. Chun Lab)


Experiment[edit]

  • Obtained PI stained mouse NPC enriched population (Yun)
  • FACs sorted PI negative population into 2ul FACS buffer in 96-well plate - could not get cells concentrated enough due to the dilution by sheath fluid
  • FACS sorted into 0.4ml facs buffer - 150,000 cells
  • Centrifuged at 1000rpm for 5 min and resuspended in 150ul
  • counted: 330,000 cells per ml (or 330 cells/ul)
  • Used 990 cells for loading (recommended was between 500-750) due to expected smaller size of the cells that may reduce capture efficiency
  • Captured only 15 cells, reloaded chip second time
  • Captured about 23 cells or 1/4 of chip - continued with RT/PCR
  • Likely cells are smaller than we expected and have very poor capture efficiency with this chip size (10-17um). In the future we may be able to increase efficiency using a smaller chip or by increasing the cell number to 2500-3000 for loading
  • Next time, it would be better to do a mass sort as quick as possible (to prevent progressive dying), spin the pool of cells down and resuspend in a very low volume (2-3x more than expected) for over 1 million per ml


FACS Sort:

File:Npc.jpg