Organoid RNA extraction protocol

1) Remove media from organoids, replace with cell recover solution* (at least 350ul/well) and

place on ice for at least 15 minutes (as many wells as desired, but should be at least 2-3).

2) Resuspend and pool each well in a 2ml Eppendorf tube (or larger centrifuge tube to

accommodate larger volumes). Centrifuge 1250 rpm, 5 min, 4°C.

3) Remove as much supernatant as possible and replace with chilled PBS.

4) Repeat steps 2 and 3 at least 2 times.

5) Resuspend organoids with 1ml Trizol reagent. Pipette up and down several times to mix and

homogenize cells.

6) Incubate room temperature for 5 min.

7) Add 0.2ml Chloroform per 1ml Trizol.

8) Secure the lid tightly and shake vigorously for 10-15 seconds.

9) Incubate for 2-3 minutes at RT.

10) Centrifuge for 15min, 12,000 x g, at 4°C.

11) Gently collect the clear-aqueous upper layer and transfer to a fresh RNAse-free 1.5 ml

Eppendorf tube. Be careful not to disturb the red-phenol-chloroform lower layer, or white

precipitate in the interphase.

12) Add 1ul of 20mg/ml RNAse free glycogen.

13) Add 0.5ml isopropanol .

14) Incubate at -20°C for at least 1 hour or overnight.

15) Centrifuge for 15 minutes, 12,000 x g, at 4°C.

16) Discard the supernatant.

17) Wash the pellet in 1ml cold 75% ethanol and centrifuge for 5 minutes, 12,000 x g, at 4°C.

18) Repeat #17.

19) Discard the supernatant with a pipette and air dry for 5-10 minutes. Do not dry entirely.

20) Resuspend in 50ul RNAse free dH2O.

21) Treat with RNAse-free DNase I using manufacturer protocols.

22) Repeat steps 5-20.

23) Quantify Final RNA concentration with a nano-drop or bioanalyzer and store at -80°C.

* Cell recovery solution is a proprietary mix that is used to degrade Matrigel. Corning Product

#354253. https://catalog2.corning.com/LifeSciences/enUS/Shopping/ProductDetails.aspx?productid=354253(Lifesciences)