Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/07/24: Difference between revisions

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==Overview==
==Major Aims==
 
<b>1. U2OS-PcTF and U2OS-TF stable cell lines <u>ChIP-PCR</u> on (1) PcTF, (2) H3K27me3, (3) H3K4me3, and (4) PolII before, during, and after PcTF expression at endogenous enhancer(s)</b>
 
Update: Cell lines are being split into plates by Dr. Haynes.
 
 
<b>2. <u>Dox dose curve</u> followed by flow cytometry and qRT-PCR on gene panel (subset); also do this for TF cell line if possible</b>
 
Update: KAH126-U2OS cells were split into two 12-well plates. Dox induction began at 5pm today.
 
 
<b>3. <u>qRT-PCR</u> experiments with TF control plasmid. Cannot select stables because PcTF is toxic. Try normalizing to PcTF levels. Include CDKN2A.</b>
 
Update:
 
 
<b>4. Another round of <u>RNA-seq</u>: (1, 2) U2OS-PcTF cells no-dox, (3, 4) U2OS-PcTF cells +dox ...and for good measure (5,6) U-2OS delta-TF (transient)</b>
 
Update: KAH126-U2OS cells were induced on Friday, and U-2OS cells were transfected with KAH132 on Friday. Visible expression in both plates (see below).
 
KAH126-U2OS, 48 hours post induction.
 
Well 1:<br>
[[Image:2016-07-24_Well_1_1_ug-mL_doxycyclin_phase_mCherry_10x.jpg | 500px]]
 
Well 2:<br>
[[Image:2016-07-24_Well_2_1_ug-mL_doxycyclin_phase_mCherry_10x.jpg | 500px]]
 
Well 3:<br>
[[Image:2016-07-24_Well_3_1_ug-mL_doxycyclin_phase_mCherry_10x.jpg | 500px]]
 
 
U-2 OS transfected with KAH132_MV2, 48 hours post transfection.
 
Well 1:<br>
[[Image:2016-07-24_Well_1_KAH132-MV2_phase_mCherry_10x.jpg | 500px]]
 
Well 2:<br>
[[Image:2016-07-24_Well_2_KAH132-MV2_phase_mCherry_10x.jpg | 500px]]
 
Well 3:<br>
[[Image:2016-07-24_Well_3_KAH132-MV2_phase_mCherry_10x.jpg | 500px]]
 


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Revision as of 10:52, 25 July 2016

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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: Cell lines are being split into plates by Dr. Haynes.


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

Update: KAH126-U2OS cells were split into two 12-well plates. Dox induction began at 5pm today.


3. qRT-PCR experiments with TF control plasmid. Cannot select stables because PcTF is toxic. Try normalizing to PcTF levels. Include CDKN2A.

Update:


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: KAH126-U2OS cells were induced on Friday, and U-2OS cells were transfected with KAH132 on Friday. Visible expression in both plates (see below).

KAH126-U2OS, 48 hours post induction.

Well 1:

Well 2:

Well 3:


U-2 OS transfected with KAH132_MV2, 48 hours post transfection.

Well 1:

Well 2:

Well 3:
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