Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/08/01
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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: Performed RNA extraction using RNeasy kit. RNA concentrations listed below.
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.
Update: Performed RNA extraction using RNeasy kit on U-2 OS samples transfected with KAH132. RNA concentrations listed below.
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.
Samples submitted to DNASU on 7/29.