Small Scale RNA Extraction
Total RNA was extracted using the GenElute Total RNA purification kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, cell pellets were resuspended in 100 ul of TE buffer containing 1 mg/ml Lysozyme, and incubated at room temperature for 5 minutes. 300 ul of buffer RL and 200 ul of 96-100% ethanol were added to the lysate before vortexing. Lysate was then applied to the spin column resin, before washing with ethanol solution. RNA was eluted in 50 ul of elution solution. Residual DNA in the RNA sample was then removed through addition of 2 units of RNase free DNase I, and incubation at 37C for 30 minutes. RNA was purified from the DNase reaction using the Monarch RNA Cleanup kit (50 ug) (New England Biolabs), following the manufacturer’s instructions. Briefly, 2 volumes of RNA binding buffer and 3 volumes of 96-100% ethanol were added to the RNA sample. The RNA was then bound to the spin column resin, before washing with ethanol solution. RNA was eluted from the column in nuclease free water, and stored at -80C. The concentration of samples was determined using a Nanodrop spectrophotometer (Thermo Fisher). Integrity of RNA samples were assessed using denaturing agarose gel electrophoresis. An equal volume of 2X RNA loading dye was added to 200 ng of total RNA, before heating to 65C for 5 minutes, and loading on a 1% agarose gel, which was run at 80 V for 40 minutes.