Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/07/24: Difference between revisions
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<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> | <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: KAH126-U2OS cells were split into two 12-well plates. Dox induction began at 5pm today. | Update: KAH126-U2OS cells were split into two 12-well plates. Dox induction began at 5pm today. Will harvest cells for flow cytometry, RNA extraction and cDNA synthesis on 7/28. | ||
Order more of the RNA extraction kit. | |||
<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> | |||
<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: don't know status of U2OS cells, need to check on that. Also need to make more KAH132_MV2 plasmid. Transforming DH5α-T cells overnight, will start liquid culture tomorrow afternoon. | Update: don't know status of U2OS cells, need to check on that. Also need to make more KAH132_MV2 plasmid. Transforming DH5α-T cells overnight, will start liquid culture tomorrow afternoon. | ||
Check for levels of probes, order more if needed. | |||
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[[Image:2016-07-24_Well_3_KAH132-MV2_phase_mCherry_10x.jpg | 500px]] | [[Image:2016-07-24_Well_3_KAH132-MV2_phase_mCherry_10x.jpg | 500px]] | ||
Will harvest two KAH126 +dox wells, two KAH126 no dox wells, and two U-2OS + KAH132-MV2 transfection wells on 7/26. Will also take photos prior to harvesting for RNA-seq. | |||
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Revision as of 14:32, 26 July 2016
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Major Aims1. 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: Dr. Haynes has ordered primers (check this). Cell lines are being split into plates by Rene and Ben. Looks like the U2OS-TF line is not growing. To do: Check U2OS-PcTF cell line and split into T-75 for Rene.
Update: KAH126-U2OS cells were split into two 12-well plates. Dox induction began at 5pm today. Will harvest cells for flow cytometry, RNA extraction and cDNA synthesis on 7/28. Order more of the RNA extraction kit.
Update: don't know status of U2OS cells, need to check on that. Also need to make more KAH132_MV2 plasmid. Transforming DH5α-T cells overnight, will start liquid culture tomorrow afternoon. Check for levels of probes, order more if needed.
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.
Will harvest two KAH126 +dox wells, two KAH126 no dox wells, and two U-2OS + KAH132-MV2 transfection wells on 7/26. Will also take photos prior to harvesting for RNA-seq.
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