TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

Media was aspirated from the well and cells were washed 2X with PBS. TriPure Trizol reagent was added to the cells and incubated at room temperature for 5 minutes. Cells were collected into a GentleMACS M tube and dissociated using the GentleMACS Dissociator (Miltenyi Bio) Program RNA01.01. Tubes were centrifuged for 3 minutes at 800RPM and the mixture was transferred to a 2ml collection tube. Chlorofom was added in a 1:5 ratio to the tripure/cell mix and tubes were inverted to mix, then incubated at room temperature for 3-5 minutes.
Aqueous phase separation was performed and the RNA-containing layer was mixed with an equal volume of 100% Isopropanol and incubated overnight at -20 degrees for precipitation. RNA was pelleted and washed with 80% ETOH, and eluted in nuclease-free water. Nucleotide concentrations were determined using Nanodrop 2000. 1µg of RNA was reverse transcribed using iScript cDNA Synthesis Kit (BioRad).
Primer sequences are shown in supplemental Table 6.

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Manufacturer protocol

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