Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/23

From OpenWetWare

Jump to: navigation, search
RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Main project page
Previous entry      Next entry

Summary

The KAH126/U2OS cells in the 6-well plate that were induced to produce PcTF-RFP back on Monday are ready for the first round of harvesting. I'll be isolating the RNA from them and storing the RNA at -80°C before sending the samples out for analysis early next week.

To be clear, I currently have two batches of KAH126/U2OS induced by doxycycline. One batch was inoculated on 1/16 and induced 1/19. I'm going to be harvesting three of the wells from that batch for RNA sequencing today. The other batch was inoculated on 1/20 and induced 1/22. I've taken fluorescence microscopy photos of both experiments.

Also need to passage the SKNSH culture from PS-β to PS-1 and the KAH126/U2OS culture from PS-4 to PS-5.



Goals

  • Passage SKNSH
  • Passage KAH126/U2OS, save some for Karmella's plates
  • Harvest RNA from KAH126/U2OS induced on Jan 19th



Protocol

TRIzol/RNEasy Spin Column Protocol

passaging mammalian cells




Notes

Observations, measurements, placeholder info etc. go here.



Results/Conclusions

Photographs of the KAH126/U2OS samples induced on 1/19 (4 days post-induction):

Well 1 Well 2

Well 3 Well 4

Well 5 (negative control) Well 6 (negative control)


Photographs of the KAH126/U2OS samples induced on 1/22 (1 day post-induction):

Well 1 (negative control) Well 2 (negative control)

Well 3 Well 4

Well 5 Well 6


Photographs of the U2OS samples transfected on 1/22 (1 day post-transfection):

Well 1 Well 2

Well 3 Well 4

Wells 3 and 4 were transfected with a plasmid made from a different batch than wells 1 & 2, thus the greatly reduced efficiency.

Well 5 (negative control) Well 6 (negative control)


RNA extraction of wells 1, 2 and 4 from the KAH126/U2OS plate induced on 1/19 (first set of images, above) was successful. RNA is in the -80°C freezer awaiting analysis.



Personal tools