Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/09/16
|cDNA prep for U-2 OS samples||Main project page|
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After harvesting U-2 OS samples over the past 2 weeks, I'll be purifying the RNA and converting into cDNA using the Superscript III first-strand synthesis kit (Life Technologies 18080-051).
This is a time course experiment, with each time point containing two biological replicates and a control.
Samples were first grown to confluency in 6-well plates, then treated with 4 µg of KAH126-MV2 plasmid and transfected following the Lipofectamine LTX protocol. After incubation for the reported time, samples were harvested, measured via flow cytometry and fluorescence microscopy, and then digested in TRIzol before being stored at -80 °C.
Samples in TRIzol were treated using the TRIzol/RNeasy protocol. Samples were then measured using the BioTek plate reader.
Measured concentrations match what's expected from the treatment schedule - less RNA will be present in samples that were treated with plasmid due to cell death and inhibited growth compared to negative controls.
Running 3 reactions (6 µg) per sample, for a total of 27 reactions. Some samples have too low of an RNA concentration to load 2 µg per reaction, so I'll be running the max 8 µL instead.
oligo(dT) Primer-RNA annealing reactions
--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.
Samples (in triplicate):
--> Aliquot 10 μL of mix into 8-tube strip