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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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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: René is culturing cells!
 
 
<b>2. <u>Dox dose curve</u> 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</b>
 
Update: RNeasy kit ordered. Cells are still growing, looking good so far. Will harvest on Thursday, 7/28.
 
 
<b>3. <u>qRT-PCR</u> 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.</b>
 
Update: Plasmid midiprep worked. Concentration is ~400 ng/µL, good 260/280 ratio. Verification image:
 
[[Image:2016-07-27_KAH132_MV2.jpg | 250px]]
 
SK-N-SH and K562 cultures expanded growing in PS-β. U-2 OS split into 6-well plates.
 
Still need to check probes.
 
 
<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: RNA extraction successful. 260/280 values all close to 2.05. Sample concentrations:
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample ID'''
| align="center" style="background:#f0f0f0;"|'''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.


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Latest revision as of 01:50, 27 September 2017

Today's project is... Main project page
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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:

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.



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