Blue:RNA-Seq Experiments:12052016

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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)
  • Isolated nuclei:
    • Removed from -80C, transferred quickly to NEB and used scalpel to mash, transferred to dounce
    • 5 strokes with loose pestle
    • 15 strokes with tight pestle -> ice 10 min
    • Filtered 50um filter
    • Centrifuged - re-filtered (30um filter) before FACS -> 5M nuclei
    • Sorted 200K for DROP-seq
    • Note: really bad clumping after centrifuging - count was only ~32K. Therefore, only enough to run Drop-seq and too clumping to run on C1
    • Froze down remaining unsorted nuclei as potential bulk control


Note: Tissue was very amendable to dissociation

Kidney Samples: Nuclei isolation for Drop-seq[edit]

Samples from Sanjay Jain's lab (Washington University):

  • Adult kidney tissue sections (40um) K1600543FBSLC
  • Isolated nuclei:
    • Removed from -80C to ice, quickly added NEB and transferred to dounce
    • 5 strokes with loose pestle -> ice 10 min
    • 15 strokes with tight pestle -> ice 10 min
    • Removed super (Filtered 50um filter) and added 1ml NEB to undissociated tissue
    • 15 strokes with tight pestle -> ice 10 min
    • Removed super (Filtered 50um filter) and added 1ml NEB to undissociated tissue
    • 15 strokes with tight pestle
    • Centrifuged combined nuclei - re-filtered (30um filter) before FACS -> 0.6M nuclei
    • Sorted 200K for DROP-seq - post-centrifuge ~125K
    • Froze down remaining unsorted nuclei as potential bulk control