Blue:RNA-Seq Experiments:11142016
Jump to navigation
Jump to search
Kidney Samples: Nuclei isolation for Drop-seq[edit]
- Samples from Andy Ransick (McMahon lab, USC) gently frozen in 1ml cryopreservative:
- Nephron Progenitor Cells 250K 15.2wk
- 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