Blue:RNA-Seq Experiments:11102016

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Revision as of 01:15, 11 November 2016 by >B1lake
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Testing Nuclei isolation protocol: Mouse 3T3 Cells

  • Obtained cells from Andrew (p30 mouse 3T3)
  • Washed cells with 1xPBS and centrifuged. Resuspended in 1ml at 1x10e6/ml
  • Used 250ul of fresh cells for nuclear lysis protocol and quantified/visualized at each step:
    • Nuclei in NEB (pre-dounce) - 6.25x10e5 (high debris, nuclei visable)
    • Nuclei in NEB (post-dounce) - 2.82x10e5 (clumpy nuclei)
    • Nuclei after 1st centrifugation - 2.85x10e5 (Clean and separated nuclei)
    • Nuclei after 2nd centrifugation - 1.65x10e5 (Clean and separated nuclei)
  • Used 250ul of fresh cells -> flash froze in liquid nitrogen -> nuclear lysis protocol:
    • Nuclei in NEB (post-dounce) - 4.05x10e5 (clumpy nuclei)
    • Nuclei after 1st centrifugation - 2.4x10e5 (Clean and separated nuclei)
    • Nuclei after 2nd centrifugation - 0.76x10e5 (Clean and separated nuclei)

Therefore, nuclear isolation protocol appears effective, maybe only a single centrifugation is needed prior to sort to ensure optimal yield. Nuclei appear ok after flash freezing, but may be more fragile since more are lost after 2nd centrifugation.

  • To ensure RNA quality after nuclear isolation, extracted RNA from 66K cells or nuclei using the Zymo MicroRNA kit and quantified RNA using Qubit:
    • Fresh Cells - 120ng/ul
    • Freshly isolated nuclei - 31.4ng/ul
    • Flash frozen isolated nuclei - 2.41ng/ul

Therefore, flash freezing the cells prior to nuclear isolation greatly affects RNA yields. The nuclear isolation methods retain the expected amount of RNA from nuclei.

  • To ensure that the nuclear extraction buffers aren't degrading the RNA, I ran 1ul or 30ng of the first two conditions on a 6% Urea gel:

File:20161110 Gel1.jpg

Nuclear RNA (lane 3) shows some LMW smearing than RNA from whole cells (lane 2), but on the whole the RNA looks not to be degraded