Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/23

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngRNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


Today we'll be harvesting the U2OS cells from the first transfection attempt. Fluorescence microscopy of the cells indicates there is little or no PcTF-RFP expression in the line, but we'll be doing a run through with flow cytometry and RNA purification regardless, as practice for future attempts.


  • Harvest U2OS cells from 1st transfection attempt
  • Flow cytometry on cells to determine gene expression levels
  • RNA purification on cells to create cDNA library (see below, Results section)


flow cytometry of mammalian cells for measuring fluorescent gene expression
RNA extraction / purification


  • PBS buffer
  • Trypsin / EDTA
  • Complete cell medium
  • FACS Buffer - 1% FBS in 1x PBS (stored at 4°C)
  • FACS tubes with strainer caps - 6 mL capacity (EMS 64750-25)
  • TRIzol / RNA purification miniprep & supplies
  • pipette tips, etc.


Overview of flow cytometry data:

20141223 flow cytometry overview.png

Negative control does not have significantly different fluorescence values than the samples (data not shown).


Since the control has the same fluorescence levels as the samples (and therefore the same gene expression levels of PcTF-mCherry), we will not be performing the RNA extraction / purification today. Instead we'll be repeating this experiment tomorrow on the second set of transfected cells.