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