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.