separating it from the top viscous layer. To avoid RNA frag-
mentation, sonication is not recommended.- We suggest to use 200μL of oligo(dT) 25 Dynabeads (bed
volume equal to approximately 15 mL of original oligo(dT)
Dynabeads) per 60 mL of cell lysate. - Care should be taken to dry the outside of the tubes before
closing them, in order to prevent leakage during the
incubation. - Keep the supernatant tube on ice until you are ready for the
second incubation round with oligo(dT) beads. Alternatively,
the supernatant may also be flash frozen in liquid nitrogen and
kept at 80 C until further use. If possible, prepare a second
batch of oligo(dT) beads to start the second round of purifica-
tions while performing the washing steps after the first round.
This may significantly decrease the total processing time. - Avoid excessive foaming of the buffer as this may lead to
retention of beads on the top of the solution. If this occurs,
pipette the top of the solution several times up and down using
a 1000-μL pipette to suspend the floating beads. - These washing steps are critical for performing shotgun prote-
omics that is incompatible with contaminating detergents
(LiDS, IGEPAL-CA630) in the sample. Ideally, the compati-
bility of the sample with downstream analyses should be dis-
cussed with the scientists performing the mass spectrometry. - After the incubation at 80C, the beads must be concentrated
and supernatant removed as quickly as possible. If processing
many tubes, perform the 2-min incubations at 80C for each
tube separately in order to avoid cooling down the sample and
reannealing of the RNA to oligo(dT) beads. - We suggest elution in 0.5–1 mL of elution buffer per 100μL
(bed volume) of oligo(dT) beads. For 60 mL of starting cell
lysate, this results in 4–8 mL of eluate after completing all three
affinity purification rounds. Typical yields can range from
0.5–5μg (low input amount) to 50–150μg of total protein
(high input amount). In our experience, some proteins (e.g.,
AGO2) are readily detected in oligo(dT) eluates by Western
analysis only after 2–3 rounds of affinity purification. - To analyze RNA in eluted samples by qRT-PCR and/or RNA-
seq, RNA can be first treated with proteinase K to digest the
proteins, followed by TRIzol extraction and further down-
stream analyses. - Alternatively, precipitate proteins with 0.25eluate volume of
trichloroacetic acid and incubation on ice for 1 h. Centrifuge at
maximum speed (16,000g, 30 min) and wash the pellet
twice with 0.20 eluate volume of cold acetone. Remove
supernatant, air-dry the pellet and resuspend it in the desired
414 Miha Milek and Markus Landthaler