3. The trypsin needs to be of sequencing grade. Regular trypsin
used for cell culture is not an appropriate substitute. Reconstitute trypsin ahead of time and aliquot as needed. The trypsin
type listed here needs to be reconstituted with needle and
gauge and then heated at 30
C for 15 min before use.
4. Dissolving PVA in water is best done with gradual heating and
mechanical stirring. This process can take several hours so it is
recommended to prepare the required 13% PVA ahead of time.
5. Streptavidin-coated beads, such as Dynabeads M-280 Streptavidin, can also be used in RNA affinity purifications with minor
adjustments to the protocol.
6. The motif discovery tool MEME is available as an online and
command-line version. For more information, please visit the
tutorial at http://meme-suite.org/doc/meme.html?man_
type¼web. HOMER is available as a command-line tool; installation instructions and tutorial are available at http://homer.
ucsd.edu/homer/motif/.
7. At least two replicates with follow-up studies is the recommended standard. When collecting lysates, please ensure that
there is enough for all replicates. In addition, because the
binding buffer only allows for 60 μL of lysates to be added, it
is better to have a concentrated sample that can be diluted with
RNase-free water.
8. Always make up a few extra mLs of buffer as a precaution.
9. While 1.5 mL tubes are suitable for this part, 2 mL tubes are
preferred as they give users some extra room to work with.
10. Usually only a small amount of sodium m-periodate is needed
to make enough of the solution. Rather than trying to accurately measure out the small amount needed, it is easier to
make up more solution than necessary and dispose of the
extra. Given the light-sensitive nature of sodium m-periodate,
do not store the solution for more than 24 h.
11. Sodium m-periodate is light sensitive, so tubes should be
placed on a rack and the rack be completely wrapped in tinfoil
and then shut in a drawer.
12. After 2 h at À80
C, the solution may freeze but should thaw
out very quickly once out of À80
C. In addition, there should
be no distinct layers; if the ethanol has formed a layer of its
own, wait for solution to thaw, and repeat steps 6 and 7.
13. If there is no dedicated 4
C centrifuge, the equipment should
be chilled down while the samples are in À80
C.
14. The best practice is to remove most of the supernatant with a
pipette and then air-dry the sample with the cap open. At the
end, there should be a small pellet at the bottom of the tube.
Using Bead-Tagged RNAs to Identify RBPs
251
used for cell culture is not an appropriate substitute. Reconstitute trypsin ahead of time and aliquot as needed. The trypsin
type listed here needs to be reconstituted with needle and
gauge and then heated at 30
C for 15 min before use.
4. Dissolving PVA in water is best done with gradual heating and
mechanical stirring. This process can take several hours so it is
recommended to prepare the required 13% PVA ahead of time.
5. Streptavidin-coated beads, such as Dynabeads M-280 Streptavidin, can also be used in RNA affinity purifications with minor
adjustments to the protocol.
6. The motif discovery tool MEME is available as an online and
command-line version. For more information, please visit the
tutorial at http://meme-suite.org/doc/meme.html?man_
type¼web. HOMER is available as a command-line tool; installation instructions and tutorial are available at http://homer.
ucsd.edu/homer/motif/.
7. At least two replicates with follow-up studies is the recommended standard. When collecting lysates, please ensure that
there is enough for all replicates. In addition, because the
binding buffer only allows for 60 μL of lysates to be added, it
is better to have a concentrated sample that can be diluted with
RNase-free water.
8. Always make up a few extra mLs of buffer as a precaution.
9. While 1.5 mL tubes are suitable for this part, 2 mL tubes are
preferred as they give users some extra room to work with.
10. Usually only a small amount of sodium m-periodate is needed
to make enough of the solution. Rather than trying to accurately measure out the small amount needed, it is easier to
make up more solution than necessary and dispose of the
extra. Given the light-sensitive nature of sodium m-periodate,
do not store the solution for more than 24 h.
11. Sodium m-periodate is light sensitive, so tubes should be
placed on a rack and the rack be completely wrapped in tinfoil
and then shut in a drawer.
12. After 2 h at À80
C, the solution may freeze but should thaw
out very quickly once out of À80
C. In addition, there should
be no distinct layers; if the ethanol has formed a layer of its
own, wait for solution to thaw, and repeat steps 6 and 7.
13. If there is no dedicated 4
C centrifuge, the equipment should
be chilled down while the samples are in À80
C.
14. The best practice is to remove most of the supernatant with a
pipette and then air-dry the sample with the cap open. At the
end, there should be a small pellet at the bottom of the tube.
Using Bead-Tagged RNAs to Identify RBPs
251