Blue:RNA-Seq Experiments:11102016
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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.
- 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:
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