15. Dissolve the total RNA pellet in 20 μL of water. Optional: Use
TapeStation and an RNA ScreenTape to analyze the integrity of
eluted RNA (RIN, RNA Integrity Number).
16. Use your preferred kit to transform the total and bound intact
RNA samples into cDNA libraries for RNA-seq. For the bound
RNA samples avoid ribosomal RNA depletion and use mild
RNA fragmentation conditions.
4 Notes
1. APEX2 is an engineered soy ascorbate peroxidase that can
oxidize biotin-phenol in the presence of hydrogen peroxide,
thus generating rapidly decaying biotin-phenoxyl radicals (t 1/
2 < 1 ms) [4]. The majority of radicals decay by reacting with
water, but some will react with APEX2-proximal proteins at
aromatic amino acids and biotinylate them. Therefore,
compartment-specific proteins can be biotinylated and isolated
by affinity chromatography by fusing localization signals to
APEX2 and targeting it to a given cellular compartment.
Although the protocol is currently designed for adherent
cells, adjustments could be made to fit cells in suspension. Cells
that are weakly adherent, such as HEK293 cells, require additional caution during the numerous washing steps to avoid loss
of cells. To employ Proximity-CLIP in other, nonmammalian
or non-cell culture, systems the following requirements must
be met: (1) Cells need to be expressing APEX2.
(2) 4-Thiouridine or 4-thiouracil needs to be taken up by
cells and metabolized into 4-thioUTP. (3) Cells need to be
accessible for UV cross-linking and for administration of BP,
hydrogen peroxide, and antioxidative quenching. Mediating
proximity biotinylation by a biotin ligase such as TurboID
[19] instead of APEX2 may be preferable in cases where a
shorter labeling radius is required, which is particularly relevant
in smaller cell systems, such as yeast and bacteria. It would also
obviate the requirement for BP, hydrogen peroxide, and
administration of quenching solution, but will add the requirement of accessibility for administration of biotin. Essentially, a
good indication that a system is amenable to Proximity-CLIP
would be the identification of successful PAR-CLIP and either
APEX2- or biotin ligase-mediated labeling experiments in the
literature.
In terms of cell density, to balance the need for high cell
number with the requirement of cells to be rapidly growing for
efficient 4SU incorporation (see Note 5) we aimed for 90%
confluence on the day of the experiment. This requires careful
calibration per cell line. We aimed to have at least ~25 Â 10
6
Proximity-CLIP
301
TapeStation and an RNA ScreenTape to analyze the integrity of
eluted RNA (RIN, RNA Integrity Number).
16. Use your preferred kit to transform the total and bound intact
RNA samples into cDNA libraries for RNA-seq. For the bound
RNA samples avoid ribosomal RNA depletion and use mild
RNA fragmentation conditions.
4 Notes
1. APEX2 is an engineered soy ascorbate peroxidase that can
oxidize biotin-phenol in the presence of hydrogen peroxide,
thus generating rapidly decaying biotin-phenoxyl radicals (t 1/
2 < 1 ms) [4]. The majority of radicals decay by reacting with
water, but some will react with APEX2-proximal proteins at
aromatic amino acids and biotinylate them. Therefore,
compartment-specific proteins can be biotinylated and isolated
by affinity chromatography by fusing localization signals to
APEX2 and targeting it to a given cellular compartment.
Although the protocol is currently designed for adherent
cells, adjustments could be made to fit cells in suspension. Cells
that are weakly adherent, such as HEK293 cells, require additional caution during the numerous washing steps to avoid loss
of cells. To employ Proximity-CLIP in other, nonmammalian
or non-cell culture, systems the following requirements must
be met: (1) Cells need to be expressing APEX2.
(2) 4-Thiouridine or 4-thiouracil needs to be taken up by
cells and metabolized into 4-thioUTP. (3) Cells need to be
accessible for UV cross-linking and for administration of BP,
hydrogen peroxide, and antioxidative quenching. Mediating
proximity biotinylation by a biotin ligase such as TurboID
[19] instead of APEX2 may be preferable in cases where a
shorter labeling radius is required, which is particularly relevant
in smaller cell systems, such as yeast and bacteria. It would also
obviate the requirement for BP, hydrogen peroxide, and
administration of quenching solution, but will add the requirement of accessibility for administration of biotin. Essentially, a
good indication that a system is amenable to Proximity-CLIP
would be the identification of successful PAR-CLIP and either
APEX2- or biotin ligase-mediated labeling experiments in the
literature.
In terms of cell density, to balance the need for high cell
number with the requirement of cells to be rapidly growing for
efficient 4SU incorporation (see Note 5) we aimed for 90%
confluence on the day of the experiment. This requires careful
calibration per cell line. We aimed to have at least ~25 Â 10
6
Proximity-CLIP
301