TITLE: High-quality RNA purification with on-column DNase treatment from tissue specimens 
AUTHORS: Magda Bletsa, Antonios Fikatas, Yiqiao Li, Sophie Gryseels, Jan Felix Drexler, Philippe Lemey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used for total RNA purification from tissue samples. It includes an intermediate on-column DNase treatment, which results in higher RNA yield and purity. This method is mainly optimised to obtain hepacivirus RNA extracts for whole-genome sequencing, but it can also be used for any viral RNA purification from samples with lower viral loads. </div></div>

[STEPS]
?. [Sample homogenisation]
Add RLT Buffer to the Precellys lysate tubes.
600 µl
?. [Sample homogenisation]
Excise a lentil-sized piece of tissue (maximum amount of  for RNAlater stabilized tissues and  for fresh or frozen tissues) and transfer it quickly to the lysate tubes. Make sure that all tissues are immersed into the RLT reagent.
20 mg
30 mg
?. [Sample homogenisation]
Place tubes on dry ice for  (or until frozen) and then thaw quickly.
?. [Sample homogenisation]
Immediately after thawing disrupt the tissues using a conventional rotor-stator homogeniser (Minilys). The recommended lysis should be performed at medium speed () for .
Centrifuge: 4000 33
?. [Sample homogenisation]
Centrifuge the lysate for  at full speed. Carefully remove the supernatant by pipetting, and transfer it to a new  Eppendorf tube.
1.5 ml
?. [RNA binding ]
Add  70% ethanol to the supernatant, and mix immediately by pipetting 5 times. DO NOT VORTEX OR CENTRIFUGE. Proceed immediately to the next step.
600 µl
?. [RNA binding ]
Transfer of the sample to an RNeasy spin column placed in a  collection tube. Centrifuge for  at If the sample is more than , change collection tubes and transfer the rest to the spin column and centrifuge again. Discard the collection tubes and replace with new ones.
700 µl
2 ml
Centrifuge: 10000 33
700 µl
?. [Sample homogenisation]
Place tubes on dry ice for  (or until frozen) and then thaw quickly.Check the results and repeat the homogenisation until no more visible fragments are present.
?. [RNA binding ]
AddBuffer RW1 to the RNeasy spin column and centrifuge for  at  to wash the spin column membrane. Change collection tubes.
350 µl
Centrifuge: 10000 33
?. [DNase treatment]
Prepare the DNase treatment master mix, as described: For each sample, add  DNase I stock solution to  Buffer RDD. Mix by gently inverting the tube, and spin down briefly. DO NOT VORTEX.
10 µl
70 µl
?. [DNase treatment]
Add the  DNase I incubation mix directly to the RNeasy spin column membrane, and place on benchtop at for .
80 µl
0 Room temperature
?. [Washing steps]
AddBuffer RW1 to the RNeasy spin column and centrifuge for  at  to wash the spin column membrane. Change collection tubes.
350 µl
Centrifuge: 10000 33
?. [Washing steps]
AddBuffer RPE to the RNeasy spin column and centrifuge for  at . Change collection tubes.
500 µl
Centrifuge: 10000 33
?. [Washing steps]
AddBuffer RPE to the RNeasy spin column and centrifuge for  at . Change collection tubes.
500 µl
Centrifuge: 10000 33
?. [Dry centrifugation]
Place the RNeasy spin column in a new collection tube and centrifuge at full speed for  If there is still much liquid passing through the column, change collection tubes and centrifuge for .
?. [Elution and storage]
Place the spin column in a new  Eppendorf tube. Add  RNase-free water directly to the spin column membrane. Incubate for  at . Then centrifuge for  at  to elute the RNA.
1.5 ml
50 µl
0 Room temperature
Centrifuge: 10000 33
?. [Elution and storage]
Repeat step 16. Use a new   tube for another elution round of .
1.5 ml
50 µl
?. [Elution and storage]
Store at -- . Optionally, you can put the column back to the corresponding collection tube and store them at --  for another future elution. Even after the 3rd or 4th elution time, there are still some RNA molecules passing through the column.
20 °C
20 °C