ࡱ> ^a]O (abjbjCC .h)e)e$[ F%4YYYmmm8d <mW*EE(mmmHHH)))))))$G,.d<)YHHHHH<)YYmm*HYmYm)H)"!N&mPcp&)'*0W*x&a/da/&&&a/Y&\HHHHHHH<)<)HHHW*HHHHa/HHHHHHHHH : Mother Enrichment Program MEP strains The MEP utilises diploid strains of a specific background made by the Gottschling lab (UCC5179 and UCC5181, JH lab strains JH969 and JH970). Genetic manipulations must be performed in both a and  backgrounds then mated (selection on -MET -LYS) to create diploids prior to use. Note that the MEP strains are LEU2 NAT HYG. Simple ageing for viability assay without enrichment Pick a colony from a fresh plate and grow for 6-8 hours in 4ml YPD or SC in a 14ml culture tube to OD 0.25-1 at 30 with shaking Dilute cells into 25ml media in a 50ml flask and grow overnight at 30 with shaking Assuming OD is 0.5, dilute 1:7,500 for YPD or 1:2,000 for SD These dilutions are for the MEP wt. If using a mutant or media with unknown growth rate, set up a few different dilutions Inoculate log phase cells into final culture at 2x104 cells/ml with 1 M -estradiol Check viability daily by plating an estimated 200 cells (10l culture diluted with 40l water) on YPD, and test the OD every day it should not exceed ~1.0 Standard Ageing with biotin labelling and purification This is written for one 125ml culture, but in reality you rarely have only one condition or time-point so be aware that this protocol is designed to be scaled up. You can handle cells for 12 conditions in a 1.5ml tube if you scale all the reagent volumes During Biotin labelling and induction it is critical that you dont lose cells when harvesting and washing. Every time that the protocol below says to spin the cells, you MUST perform the two step spin described in the Why wont my cells pellet? section of the Harvesting Yeast protocol. Pick a colony from a fresh plate and grow 6-8 hours in 4ml media (YPD, SD or whatever you will use for ageing) in a 14ml tube to OD 0.25-1 at 30 with shaking Dilute cells into 25ml media in a 50ml flask1 and grow overnight at 30 with shaking Assuming OD is 0.5, dilute 1:7,500 for YPD or 1:2,000 for SD These dilutions are for the MEP wt. If using a mutant or media with unknown growth rate, set up a few different dilutions Next day the OD should be 0.2-0.8. Harvest 1.25x106 cells per ageing culture in a 1.5ml tube, using the Why wont my cells pellet? harvest method If you need more than 1.5ml culture, just spin down 1.5ml, remove most of the supernatant and add more cells. Wash cells twice with 125l PBS, spinning 15s at top speed Re-suspend cells in 62.5l per 1.25x106 cells of 3mg/ml biotin-NHS in PBS and incubate on wheel for 30 min at room temperature Biotin-NHS powder is stored in 1 and 1.5mg aliquots. Combine as many as you need to make just enough 3mg/ml Biotin-NHS solution for your experiment. You cannot store this solution, make it immediately before use. The actual concentration does not need to be very accurate - 25% is fine Wash cells 1x with 100l PBS, spinning 15s at top speed Re-suspend the cells in 62.5l PBS per 1.25x106 and inoculate 62.5l in each ageing culture as follows: 24/48 hour: Inoculate in 125 ml media in 250ml flask. Incubate at 30 with shaking for 2 hours. This incubation MUST be 1.25-1.5x the doubling time of the given strain in the given media. You can work this out based on the starting and finishing density of your pre-culture. Then add 125 l 1mM -estradiol (CARE!), and any other drugs needed. 7.5 hour: Inoculate in 15 ml media in 50ml flask. Incubate at 30 with shaking for 2 hrs (or longer as above), add 15 l 1mM -estradiol (CARE!). Check the OD every day  should not exceed 1.0 (or at least not by much) Harvest cells for DNA/RNA/protein/flow: spin down 125ml in a 50ml falcon tube (ie: spin 50ml, pour off, add another 50ml, etc), re-suspend in 70% ethanol to store at -80( Harvesting cells for ChIP or IF or flow: Fix with 1% formaldehyde (use Thermo 11586711 for ChIP, Sigma F8775 for IF or FACS), neutralise by adding glycine to 125mM, wash and freeze as pellets of cells from 125ml culture on N2 Purification This protocol is designed for multiple samples. We routinely perform up to 12 simultaneously for DNA or IF (using 3 QuadroMACS magnets), but this can be quite slow. For RNA you need to be quick or you do get some degradation, so initially do batches of up to 4 simultaneously and once up to speed do 6-8 at most. Put 1.3ml Percoll gradient mix in two 2ml tubes per sample (use a single gradient if the cell pellet is small eg: for 7.5 hour samples), vortex and spin 15min at 16kg, 4(. Formaldehyde fixed cell pellets: thaw directly in 0.5ml PBSE Ethanol fixed cells: run under tepid water for a few minutes if frozen, spin 1 min at 4,000rpm, pour off ethanol, wash with 25ml cold PBSE, spin 1 min at 4,000rpm, pour off and re-suspend pellet in the residual PBSE (~0.5ml) Layer the cells carefully on the top of the two gradients, this is easiest when holding the tube at an angle Spin the gradients 2000g, 4min at 4( - they should separate into a pellet about half way down and a thick band near the top Meanwhile, if you have log phase control cells re-suspend these in PBSE and keep them at the same temperatures as the aged cells during the purification. Carefully remove the top of the gradient, getting rid of all the thick band of cell debris Add 1ml PBSE to the rest of the gradient, vortex and spin 1 min at 5000g at 4 the cells should all pellet; if not remove most of the liquid, add more PBS vortex and re-spin. Remove the supernatant, re-suspend the cells from each gradient in 0.5 ml PBSE, and combine to give 1 ml cell suspension (you normally have ~50% of the cell number you started with) Add 25l streptavidin MACS beads to 1ml percoll-purified cells, and incubate 15 min on a wheel at room temperature OR 5 MIN ONLY FOR RNA! Work in the cold room from here onwards if you need RNA or protein: Equilibrate a MACS LS column on QuadroMACS magnet with 1ml PBSE at 4( Load the cell suspension into the column, let it flow through under gravity (1-2 min) Wash the column with 1ml PBSE, let it flow through under gravity (1-2 min) Elute the cells by removing from the magnet, adding 1ml PBSE and forcing through into a 1.5ml tube using the syringe Re-equilibrate the column with 0.2-0.5ml PBSE, re-load the cells and repeat the washing and purification. Do three rounds of this to get nicely purified cells. Set aside 50l cells for rapid biotin staining, add 1l of 10% Triton X100 to the rest then pellet (spin 30s at top speed), remove supernatant and freeze on N2 or directly process as convenient. Rapid biotin staining To each cell sample add: 240l PBS (275l for unsorted cells) 9l 10% triton X100 0.3l 2mg/ml streptavidin 594 0.6l 1mg/ml Alexa 488-WGA 0.1l 1.5 mg/ml DAPI solution Mix and incubate 15min at room temperature (much longer is no problem) Spin cells 10s top speed, wash 1x with 300l PBS + 0.01% Triton-X100 Re-suspend cells in 7l Vectashield and examine using fluorescence and phase contrast. >80% of purified cells should be brightly stained in 594 channel. The WGA allows bud scar counting. Column re-use MACS columns are expensive and we re-use ours many times without issue. After use, remove the column from the magnet and flush twice with ~5ml of water, forcing it through with the syringe. Then flush with 100% ethanol (put in the syringe a few times to ensure all the ethanol is gone) and dry for ~ 1 hours in a 65( oven. Store the dried columns at room temperature. If the column goes rusty throw it away, but this should not happen and indicates a problem with the washing procedure. Before re-use, put the column on the magnet, add ~5ml PBSE, insert the plunger a little way repeatedly to expel bubbles of air from the column. Let the rest of the PBSE flow through by gravity Solutions and reagents We use filtered YP or synthetic media for all ageing experiments to remove variability from autoclaving PBSE: cold PBS + 2mM EDTA -estradiol stock is 1mM in ethanol, stored at -80( in 1ml aliquots. Sigma cat # E2758 Estradiol is highly bio-active, handle with care. In our lab, pregnant women do not handle the stock solution. Handle the powder only in the hood, put a small amount in a pre-weighed 50ml tube, weigh this then add the required amount of ethanol. Seal well then sonicate 10s on low in a Bioruptor to break up the crystals then aliquot in screw cap tubes. Because it is stored in ethanol the solution tends to leach out around the top of the tube, so change gloves after opening/closing the stock. Biotin-NHS: AKA Biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-NHS-LC-Biotin, Pierce cat #21335 (100mg)). Buy 100mg bottle, warm to room temperature for 2 hours before opening then split into 1 and 1.5mg aliquots in 1.5ml tubes and freeze. The protocol is forgiving and the weight of the aliquots can be +/- .25mg. Can also be purchased from Sigma but this is less soluble in our hands. Streptavidin MACS beads: MILTENYI 130-048-101 Percoll gradient mix: 45 ml percoll 1.6 ml 5M NaCl 3.4 ml water Notes on the MEP strain We obtained MEP haploids and diploid from the Gottschling lab when it was still at the Fred Hutch. 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The a haploid is pink due to ADE2 deletion, but throws up white colonies at quite a rate. These are still ADE- but we avoid them. The MEP system seems to work much better at the UBC9 than the CDC20 locus  The high volume here is helpful to ensure that cells are growing very happily at induction  Do not plate more and dont plate undiluted culture the residual estradiol transferred to the plate can impact colony growth. If you need to plate more, you must spin and wash ~1ml culture  Remember that although the cell density increases, the number of live cells does not the density increase comes from inviable daughters. Note also that daughters are not metabolically dead and will consume nutrients, so keep the OD low. If the OD suddenly increases dramatically or you have more colonies on the plates than you started with then the cells have escaped the MEP system.  If you are using SD media you can double the number of cells per ageing culture, but label with 2x biotin-NHS volume.  You often set cultures for 2-3 ageing time-points which are labelled and washed in a single tube.  This volume of wash is fine up to at least 5x106 cells  Biotin-NHS from Pierce dissolves immediately in PBS. Other brands we have used need vortexing. When aliquoting Biotin powder, leave the vial at room temperature for ~1h beforehand, so that the condensation can evaporate.  Ethanol fix is easier and allows samples to be used for DNA/RNA/protein as well as flow, but formaldehyde fix is better for weak fluorophores  Alternatively, use 0.9ml Percoll and 100l 10x PBS, vortex well. This is our standard mix for RNA and ChIP. It makes little difference to gradients, we have also got decent RNA from the standard mix  If cell OD at harvest was low (<0.4) then use only one gradient  Dont push the plunger in too far or you will suck air back into the column when you pull it out again. 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