Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/07/27

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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: René is culturing cells!

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, looking good so far. 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: Plasmid midiprep worked. Concentration is ~400 ng/µL, good 260/280 ratio. Verification image:

2016-07-27 KAH132 MV2.jpg

SK-N-SH and K562 cultures expanded growing in PS-β. U-2 OS split into 6-well plates.

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: RNA extraction successful. 260/280 values all close to 2.05. Sample concentrations:

Sample ID Concentration (ng/µL)
U2OS-PcTF +dox 1 404
U2OS-PcTF +dox 2 449
U2OS-PcTF +dox 3 350
U2OS-PcTF no dox 1 555
U2OS-PcTF no dox 2 454
U2OS-PcTF no dox 3 412
U2OS +KAH132 1 564
U2OS +KAH132 2 519
U2OS +KAH132 3 555

Will pick 2 from each sample type to submit for RNA-Seq to DNASU.