Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/08/01

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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)

Nothing new.


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: Performed RNA extraction using RNeasy kit. RNA concentrations listed below.

Sample ID ng/µL 260/280
1.1 39.122 1.956
1.2 52.618 1.953
1.3 35.561 1.933
6.1 37.135 1.959
6.2 34.885 1.798
6.3 46.068 1.893
7.1 54.586 1.86
7.2 35.873 1.969
7.3 40.628 1.911
8.1 47.349 2.017
8.2 61.411 1.99
8.3 82.889 2.058

New plan: since RNA concentrations are very low, going to perform a test cDNA synthesis on samples 1.1-1.3, followed by qPCR on target genes. If signal is good, will proceed with cDNA synthesis and qPCR on remaining samples. Do cDNA synthesis in triplicate batches on each sample (8 µL of RNA per treatment), then pool reactions together at the end. For qPCR, dilute cDNA 1:2.

Meanwhile, start a new set of two 6-well plates of U2OS-PcTF and when sufficiently high cell density, induce with dox at 1 µg/mL, 31 ng/mL, 16 ng/mL, and 0 ng/mL (control) in triplicate (12 wells total) for 4 days. This will give us enough material for a higher-yield RNA extraction next week if needed.


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: Performed RNA extraction using RNeasy kit on U-2 OS samples transfected with KAH132. RNA concentrations listed below.

Sample ID ng/µL 260/280
U2OS KAH132 24hr rep1 117.411 1.932
U2OS KAH132 24hr rep2 137.561 1.926
U2OS KAH132 48hr rep1 146.72 2.045
U2OS KAH132 48hr rep2 119.495 2.006
U2OS KAH132 72hr rep1 280.054 2.026
U2OS KAH132 72hr rep2 339.671 2.048

Transfected SK-N-SH wells with KAH132. Will take pictures, flow cytometry, and harvest on 8/2-8/4.

Still need to check qPCR 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)

Samples submitted to DNASU on 7/29.




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