Blue:RNA-Seq Experiments:11102016

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Testing Nuclei isolation protocol: Mouse 3T3 Cells[edit]

  • 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

Nuclei isolation protocol used[edit]

Nuclei isolation from Cells

  1. Prepare cells:
    1. Wash with PBS and centrifuge 300g 5min
    2. Resuspend cells to 1x106 per ml in PBS
  2. Dounce with sucrose-buffered extraction buffer (RNAse-free)
    1. Wash dounce in RNaseAway or RNaseZap, rinse with Milli-Q water
    2. Add 1ml of NEB containing 1ul DAPI (5mg/ml) to dounce and chill on ice
    3. Add 250ul cells to 1 mL ice cold NEB buffer - incubate 10 minutes on ice
    4. Gently dounce sample on ice:
      1. 5 strokes with loose pestle A
      2. ~10 strokes with pestle B (minimize bubble formation)
    5. Pass debris through 30-µm CellTrics filter to 15-mL conical tube
    6. Wash dounce with 1 mL NEB buffer, and filter this into the same tube
    7. Bring up to 10 ml with PBSE
    8. Pellet nuclei: 900g, 10 min.
    9. Resuspend pellet in 1ml PBSE+1%BSA. Add 9ml PBSE.
    10. Pellet nuclei: 900g, 10 min.
    11. Resuspend pellet in 1ml PBSE+1%BSA.