1 Collect EBs and gently pellet in a tube, by microcentrifugation at *1000rpm for 15 s. Carefully remove as much medium as possible.

2 Add 100 ml of Trizol to the sample, mix well, and incubate for 5 min at r.t.a

3 Add 20 ml chloroform per 100 ml Trizol suspension, and vortex well.

4 Centrifuge the sample at 12 000 g for 12-15 min, preferably at 4 °C.

5 Transfer the aqueous phase to a fresh microcentrifuge tube, adding a half volume of isopropanol (i.e. 50 ml per 100 ml starting Trizol); mix well, and incubate for 10 min at r.t. If using very limited amounts of RNA (1-20 EBs) it is suggested that RNase-free glycogen be added (4-5 mg).b

6 Centrifuge the sample at 12 000 g for 10 min, preferably at 4 °C, to pellet RNA.

7 Remove the supernatant carefully, and wash the RNA pellet once with 75% ethanol.

8 Centrifuge the sample for 5 min, preferably at 4 °C, and remove all ethanol. (A second, short centrifugation may be helpful.) Air-dry the pellet for * 10 min at r.t.

9 Resuspend the RNA pellet in 50 ml RNAse-free water, and rapidly freeze on dry ice. If more than 106 cells were in the starting sample, use 100 ml RNAse-free water/106 cells; for less than 105 cells (as in a single EB), either resuspend in 20 ml water or use the pellet directly in the next stage.

10 If required, and if there is a sufficient quantity, assess RNA quality by gel electrophoresis (or equivalent). Alternatively, and for cDNA applications, go directly to the DNasel treatment (Protocol 6).

a At this stage the sample can be stored at - 20 °C to - 70 °C.

bDo not add too much glycogen as it may inhibit cDNA synthesis.

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