- Pass the lysate with a syringe several times through a 23- and
26-G needle (seeNote 4). Afterward, save 0.2 mL of input
lysate for downstream analyses (e.g., Western analysis and RNA
extraction) and store it at 80 C. - Pre-wash the magnetic oligo(dT) beads in 5 mL of lysis/bind-
ing buffer in a 15 mL Falcon tube and concentrate them on a
magnet. Afterward, resuspend beads into the cell extract by
first taking them up in 1 mL of the prepared lysate and trans-
ferring them to the tube containing the remaining lysate (see
Note 5). - Incubate for 1 h at room temperature on a rotating wheel (see
Note 6). - Concentrate beads on magnet. The initial concentration of the
beads on the magnet may take from 10 min to 1 h. - Carefully collect supernatant and place it into a new tube for
additional rounds of oligo(dT) precipitation (seeNote 7). - Wash beads two times in one sample volume of room-
temperature lysis/binding buffer. For each washing step, rotate
the tubes 2–5 min on a rotating wheel at room temperature.
Afterward, concentrate the beads on magnet and carefully
remove the supernatant (seeNote 8). - Wash beads two times in one sample volume of room-
temperature washing buffer containing 0.5 mM DTT and
complete EDTA-free protease inhibitor cocktail. For each
washing step, rotate the sample 2–5 min on a rotating wheel
at room temperature. Afterward, concentrate the beads on
magnet and carefully remove the supernatant. - For the third washing step, resuspend beads in 1 mL of washing
buffer and transfer to a fresh 1.5-mL tube. Afterward, concen-
trate the beads on magnet and remove the supernatant. - Wash beads once with 1 mL of ice-cold low-salt elution buffer
to remove traces of IGEPAL CA-630 (seeNote 9). Concen-
trate beads with magnet and remove supernatant. - Resuspend beads in 0.5–1 mL of low-salt elution buffer and
incubate them for 2 min at 80C. Remove supernatant into a
new precooled 15-mL falcon tube and place it on ice as quickly
as possible. Pool the eluates from different aliquots. Once the
eluate is removed, immediately cool down the beads by placing
them on ice (seeNote 10). - Repeat the elutionstep 11once more.
- Perform several additional rounds of oligo(dT) affinity purifi-
cations. We typically perform two additional rounds with only
one elution per round for a total of three affinity purifications
(seeNote 11).
410 Miha Milek and Markus Landthaler