Algal culture harvest and RNA extraction for RNA - Seq
Use sterile techniques to harvest 800 ml of culture , and divide into 4 250 ml centrifuge bottles with 200 ml each .
Spin the centrifuge bottles at 5000 g for 10 min .
Carefully decant the supernatant .
Use 0.5 ml of RNALater to resuspend the pellet and transfer it to a microcentrifuge tube .
Use another 0.25 ml of RNALater to wash the bottle and obtain any remaining cells .
Transfer this 0.25 ml into the same microcentrifuge tube .
Depending on whether you want pseudo - replicates , you can keep the 4 harvested pellet separate or mixed .
Store the harvested cells at or below - 20oC until ready for RNA extraction .
Before RNA extraction , the RNALater needs to be removed .
Thaw samples if they are frozen .
Centrifuge samples at > 10 k g for 10 min at 4oC .
RNALater sometimes affects the buoyancy of the cells , and make them a bit harder to pellet .
If you find that the cells are not pelleting , spin harder for a longer time .
Decant supernatent with RNALater .
Extract RNA using a RNeasy Plant Mini Kit ( Qiagen ) following the manufacturer's instructions .
Remove any DNA from the extracted samples using DNase ( Sigma ) , following manufacturer's instructions .
Clean up the total RNA using a RNA Clean & Concentrator kit ( Zymo ) , following the manufacturer's instructions .
Quantify the RNA using any preferred method , e . g . Qubit fluorometer .
Store the RNA at - 80oC .
It may be ideal to aliquot some RNA out so that you don't need to thaw the entire RNA sample if you need some for any reason
