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Blue:RNA-Seq Experiments:11292016
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===PDX Single-Cell RNA Seq=== '''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
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