Blue:RNA-Seq Experiments:11292016

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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 11/21/2016
  • Used Papain Dissociation System (Worthington Enzyme Manual) to dissociate tissue to single cells

Goal: to use RNAlater to preserve the RNA prior to cell dissociation

  • Modifications to protocol:
    • Washed the tissue with EBSS (500ul) before adding papain solution.
    • Minced/chopped tissue using scalpel in papain solution
    • Dissociated for 30min in rack on vortexer (lowest setting) at 37C
    • Triturated with p1000 (10x)
  • After discontinuous gradient - few cells were detected
    • Re-centrifuged papain supernatant at 400g and repeated discontinuous gradient - again few cells detected
    • Re-centrifuged both discontinuous gradient supernatants at 400g for 5 min - a lot of debris and low number of round single cells
    • Stained with dapi and filtered with 30um filter
    • Debris stained with dapi and it was difficult to see clear single intact cells - large jagged clumps that could not be run on the C1
    • Centrifuged and stored pellet at -80C for possible RNA extraction if needed.

Outcome: Doesn't look possible to dissociate clean single cells from RNAlater treated flash frozen tissue