My embryoid bodies are very tough, it is very difficult to break down these cell aggregates and extract RNA just with pipetting. Does any one have a better idea?
First of all. DEPC-treated containers (tubes) with gloves are essential. Wash collected EBs in DEPC-PBS by spinning down 2x. EB cell braking in lysis buffer with repeated (3x) freezing in LN2 and thawing. Lysis buffer to lyse cells take time unlike recommended instruction. Give more time on rocker, ~30min at fast rocker. Then proceed RNA isolation. You could use collagenase incubation for 30 min in CO2 incubator, followed by T-E sol (1/5 dilution) to loosen the EB cells if they are too big or differentiated further.