Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/18

RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines

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Summary The transfection from earlier in the week had a very low efficiency, but we're going to expand that cell line just in case, transferring the cells into 10cm petri dishes. Meanwhile, we'll also be setting up another batch of U2OS cells for transfection by advancing them to another 6 well plate, as well as passaging the line from PS-1 to PS-2. Lastly, we'll be using the PS-1 backup flask to create more frozen stock of U2OS. Goals Expand U2OS PS-1 for 2nd transfection attempt Expand previously transfected U2OS cells into 10cm petri dishes Prepare frozen stocks of U2OS cells from PS-1 backup flask Protocol passaging adherent cells - for passaging cells from PS-1 to PS-2 cryopreservation of mammalian cell lines - for preparing frozen stock from PS-1 backup flask To advance the PS-1 cells to 6-well plate: Prepare cells in PS-1 flask for passaging, as above There's approximately 1E6 cells/mL in the T-75 flasks, and we want to put 2E5 cells into 4mL of medium in each well. Therefore, prepare 26.6mL of 10% FBS P/S medium in a 50mL tube and add 1.4mL of trypsinized cells to the tube, for a total volume of 28mL (20x dilution) Pipet 4mL of the diluted cells into each well, with 4mL left over as a safeguard Label the well plate with initials, cell line, date and passage number and place in the incubator Discard remaining liquid waste via aspiration To advance the transfected U2OS cells from 6-well plates to 10cm petri dishes: Warm up: PBS, trypsin buffer, U2OS medium (10%FBS, 1%p/s) Harvest DNA-positive cells from 6-well plates: aspirate off medium, wash with 0.5 mL PBS, trypsinize with 0.5 mL trypsin/EDTA Label 3 10cm round TC-treated sterile petri dishes. Transfer 6 mL medium into each. Collect trypsinized cells from each small well: use 3.5 mL medium, pipette up and down to make an even cell suspension. Transfer into the 6mL medium in one of the 10 cm plates. Spread cells by moving plate up/down/back/forth. Repeat for two remaining samples. Put plates in incubator. Check once a day for RFP. Materials List materials here, to be gathered before the experiment Consumables such as media, antibiotics, DNA, kits etc. Notes Observations, measurements, placeholder info etc. go here Results/Conclusions Transfected U2OS cells (and one negative control) are in 10cm petri dishes in the top incubator, third shelf from the top. A T-75 flask containing U2OS cells in PS-2 is in the top incubator, third shelf from the top. A 6-well plate containing U2OS cells prior to transfection is in the top incubator, third shelf from the top. Five 2mL cryo vials containing U2OS cells in FBS/DMSO medium are in the -80 freezer, undergoing slow cooldown. They will be ready to be transferred to the -150 freezer by Saturday, Dec 20th. David Benjamin Nyer 15:31, 18 December 2014 (EST):