Blue:RNA-Seq Experiments:11142016

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

  • Samples from Andy Ransick (McMahon lab, USC) gently frozen in 1ml cryopreservative:
  1. Nephron Progenitor Cells 250K 15.2wk
  2. Kidney Cortex (dissociated cells) 10M 16.5wk
  • Checked for live cells using trypan-blue count before isolating nuclei
    • Cortex - 32% live cells (post-centrifuge) - ~2.5M total
    • Nephron progenitors - 16% live (pre-centrifuge) - 160K total
  • Isolated nuclei using NEB and dounce and a single centrifugation step
  • Sorted on FACS AriaII (SCRM) - nuclei looked very clean with little debris. Sorted G1-S-G2/M populations.
    • 200K for Cortex
    • 38K for the progenitors (used all of the sample - therfore starting with 250K cells is not enough for Drop-seq)
  • Centrifuged all nuclei (900g 10min) and resuspended in 100ul of 1:10 dilution of PBSE/1%BSA in 1xPBS (for 0.01%BSA) and transferred to Song for Drop-seq
  • Experiment documented here: 20161114