Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/07/26

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Major Aims

1. U2OS-PcTF and U2OS-TF stable cell lines ChIP-PCR on (1) PcTF, (2) H3K27me3, (3) H3K4me3, and (4) PolII before, during, and after PcTF expression at endogenous enhancer(s)

Update: Nothing new since 7/24. Two T-75 flasks are growing for René's work and my own.


2. Dox dose curve followed by flow cytometry and qRT-PCR on gene panel (subset of 8 genes + GAPDH + PcTF); also do this for TF cell line if possible

Update: RNeasy kit ordered. Cells are still growing. Will harvest on Thursday, 7/28.


3. qRT-PCR experiments with TF control plasmid, on U-2 OS, SK-N-SH and K562 cell lines. Cannot select stables because PcTF is toxic. Try normalizing to PcTF levels. Include CDKN2A.

Update: transformation worked (hundreds of colonies) but plasmid midiprep failed. Maybe picked wrong colony? Trying again tonight + tomorrow.

SK-N-SH and K562 cultures expanded from PS-α to PS-β.

Still need to check probes.


4. Another round of RNA-seq: (1, 2) U2OS-PcTF cells no-dox, (3, 4) U2OS-PcTF cells +dox ...and for good measure (5,6) U-2OS delta-TF (transient)

Update: fluorescence microscopy images taken again of the samples. Cells were harvested by immersion in TRIzol, then stored at -80 C. Will perform RNeasy preps tomorrow in preparation for RNA-seq.

Well 1 (U2OS-PcTF +dox, post 96 hr):
2016-07-26 Well 1 1 ug-mL doxycyclin phase mCherry 10x.jpg


Well 2 (U2OS-PcTF +dox, post 96 hr):
2016-07-26 Well 2 1 ug-mL doxycyclin phase mCherry 10x.jpg


Well 3 (U2OS-PcTF +dox, post 96 hr):
2016-07-26 Well 3 1 ug-mL doxycyclin phase mCherry 10x.jpg


Well 1 (U2OS ΔTF (transient), post 96 hr):
2016-07-26 Well 1 KAH132-MV2 phase mCherry 10x.jpg


Well 2 (U2OS ΔTF (transient), post 96 hr):
2016-07-26 Well 2 KAH132-MV2 phase mCherry 10x.jpg


Well 3 (U2OS ΔTF (transient), post 96 hr):
2016-07-26 Well 3 KAH132-MV2 phase mCherry 10x.jpg


No photos taken of U2OS-PcTF without dox treatment.