RNA Isolation from Viral-infected Chlorella NC64A
Grow NC64A to mid-log phase.
Concentrate to 1 x 108 cells/ml.
Use 30 ml concentrated cells per time point (3 x 109 cells~91 mg cells wet weight).
Infect with virus at m.o.i.=5 for appropriate time.
Harvest infected cells by centrifugation at 5000 rpm, 5 min., 4°C.
Discard supernatant and flash freeze pellet in liquid nitrogen.
Store at -80°C until ready to proceed to step #8.
Take 1 to 2 samples from the freezer at a time (keep on ice), break pellets loose with spatula, and add 1 ml TRIZOL reagent to each tube.
Work as quickly as possible to prevent RNA degradation.
Break pellets up with spatula and vortexing.
Pipet into 2 ml microcentrifuge tube(s) containing 250 mg of 0.25-0.30 mm baked glass beads.
Immediately vortex at highest speed 5 min., 4°C.
Place at -80°C overnight.
Repeat steps #8-12 for the remaining samples.
Thaw samples and vortex another 5 minutes.
Add 250 µl chloroform to samples, then vortex briefly.
Let stand at room temp. 3 min.
Centrifuge samples at 12,000 x g, 15 min., 4°C.
Withdraw the RNA-containing aqueous upper phase and deliver to fresh microcentrifuge tube.
Add 500 µl isopropanol.
Let stand 10 min. at room temp.
Centrifuge at 12,000 x g, 10 min., 4°C.
Discard supernatant.
Wash pellet with 75% EtOH with vortexing.
Centrifuge 12,000 x g, 5 min., 4°C.
Dry pellets in vacuum dessicator 5-10 min.
DO NOT DRY COMPLETELY.
DNAse each sample by resuspending each pellet in 250 µl dd-H20.
Add 250 µl of 2X MOPS, 20mM MgCl2 (2X), and 100-200 U RNAse-free DNAse I. 
Incubate 1-2 h on ice.
Extract with equal volume of phenol:chloroform (500 µl).
Centrifuge at 12,000 x g, 5 min., 4°C.
Withdraw aqueous phase, add NaOAc to 0.3 M.   
Add 1 ml isopropanol.
Let stand 10 min., room temp.
Centrifuge up to 12,000 x g, 10 min., 4°C.
Discard supernatant.
Wash pellet with 75 % EtOH with vortexing.
Centrifuge 12,000 x g, 5 min., 4°C.
Dry pellets in vacuum dessicator 5-10 min.
Do not dry completely.
Resuspend pellets in 40-50 µl water.
