Blue:RNA-Seq Experiments:11302016

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PDX Single-Cell RNA Seq[edit]

Meduloblastoma tissue (PDX) from Weschsler-Reya Lab

  • Tissue was flash frozen on 10/19/2016 (3 separate chunks)
  • Transferred 1 chunk to -20C RNAlater ICE and stored at -20C on 11/29/2016 (i.e. overnight)
  • Isolated nuclei:
    • Removed from -80C, washed briefly with PBS, coarsely chopped using scalpel (petri dish)
    • Transfered to NEB in dounce on ice
    • 5 strokes with loose pestle -> ice 10 min
    • 5 strokes with loose pestle
    • 15-20 strokes with tight pestle - note: very difficult to dissociate - a lot of DAPI+ debris and jagged nuclei/tissue clumps. Clean intact nuclei are sparce.
    • Filtered 50um filter
    • Centrifuged - re-filtered (30um filter) before FACS -> sorted ~70K for DROP-seq
    • Froze down remaining unsorted nuclei as potential bulk control


Note: RNAlater-Ice seems to make the tissue tougher to dissociate or the cell membrane to dissolve... even worse than kidney tissue treated with regular RNAlater, although that was only for 6hrs