Blue:RNA-Seq Experiments:11102016
Jump to navigation
Jump to search
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. Flash freezing causes more loss after 2nd centrifugation
- 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