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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|400px]] ''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== Nuclei isolation from Cells #Prepare cells: ##Wash with PBS and centrifuge 300g 5min ##Resuspend cells to 1x106 per ml in PBS #Dounce with sucrose-buffered extraction buffer (RNAse-free) ##Wash dounce in RNaseAway or RNaseZap, rinse with Milli-Q water ##Add 1ml of NEB containing 1ul DAPI (5mg/ml) to dounce and chill on ice ##Add 250ul cells to 1 mL ice cold NEB buffer - incubate 10 minutes on ice ##Gently dounce sample on ice: ###5 strokes with loose pestle A ###~10 strokes with pestle B (minimize bubble formation) ##Pass debris through 30-Β΅m CellTrics filter to 15-mL conical tube ##Wash dounce with 1 mL NEB buffer, and filter this into the same tube ##Bring up to 10 ml with PBSE ##Pellet nuclei: 900g, 10 min. ##Resuspend pellet in 1ml PBSE+1%BSA. Add 9ml PBSE. ##Pellet nuclei: 900g, 10 min. ##Resuspend pellet in 1ml PBSE+1%BSA.
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