radioactive waste (Subheading 3.6, step 10), pinched into the
gel using the pipette tip. This will enable alignment of the gel
with a printout of the autoradiograph. Wrap the gel in Saran
wrap and expose a pre-blanked phosphoimager screen to the
gel in a dedicated cassette at À20
C to avoid diffusion of the
nucleic acids in the gel. Print the image in its original size and
position the gel above the printed paper by aligning the signal
and blue color of the radioactive waste. Cut gel sections that
contain the required RNAs and collect them into gel breaker
tubes.
4. Pippin Prep and 3% Pippin gel cassettes (Sage Science) are used
for agarose gel size separation and extraction of the amplified
sRNA cDNA sequencing library. This step is necessary because
directly ligated 3
0 and 5
0 adapters (“empty library”) are a common side reaction that can overwhelm the library during PCR.
If Pippin Prep is not available it is possible to use standard 3%
agarose gel electrophoresis to size-separate the two PCR products. An alternative to gel extraction of the amplified library
would be to follow the following steps: (1) Run the gel at 90 V
for ~90 min. (2) Position the gel on a UV imager and manually
carve a well right below the band corresponding to the library.
(3) Return the gel to the running apparatus, lower the buffer
height so it is just below the top of the gel, empty the liquid off
the carved well, and fill it with clean running buffer. (4) Continue to run the gel for 5 min while every 1 min collecting the
buffer from the well into a fresh tube and quickly replacing it
with fresh buffer. (5) Reimage the gel to validate that the
library band no longer appears and use TapeStation and a
D1000 ScreenTape to assess in which of the tubes the library
eluted, and to measure its concentration and average length.
5. Nascent RNAs incorporate 4SU during transcription; therefore
in most cases overcrowding of cells should be avoided to maintain a metabolically active culture. The 16-h labeling time
window can be increased for slow-growing cells, or decreased
for labeling of transient events, as required.
6. Fluorescence microscopy imaging should be performed
according to the standard procedures applied per lab, cells,
and experimental system. It is essential to image both the
APEX2-fusion protein and biotinylated proteins in all conditions (labeled and ÀBP, Àhydrogen peroxide, and ÀAPEX2
controls). Note that immunological detection of the APEX2fusion protein may vary after labeling due to self-biotinylation.
Biotinylated proteins can be labeled by fluorophore-coupled
neutravidin or an alternative (we use Alexa-Fluor-647-coupled
neutravidin). The main goals of imaging are (1) to confirm that
APEX2 is expressed and localized as expected; (2) to confirm
Proximity-CLIP
303
gel using the pipette tip. This will enable alignment of the gel
with a printout of the autoradiograph. Wrap the gel in Saran
wrap and expose a pre-blanked phosphoimager screen to the
gel in a dedicated cassette at À20
C to avoid diffusion of the
nucleic acids in the gel. Print the image in its original size and
position the gel above the printed paper by aligning the signal
and blue color of the radioactive waste. Cut gel sections that
contain the required RNAs and collect them into gel breaker
tubes.
4. Pippin Prep and 3% Pippin gel cassettes (Sage Science) are used
for agarose gel size separation and extraction of the amplified
sRNA cDNA sequencing library. This step is necessary because
directly ligated 3
0 and 5
0 adapters (“empty library”) are a common side reaction that can overwhelm the library during PCR.
If Pippin Prep is not available it is possible to use standard 3%
agarose gel electrophoresis to size-separate the two PCR products. An alternative to gel extraction of the amplified library
would be to follow the following steps: (1) Run the gel at 90 V
for ~90 min. (2) Position the gel on a UV imager and manually
carve a well right below the band corresponding to the library.
(3) Return the gel to the running apparatus, lower the buffer
height so it is just below the top of the gel, empty the liquid off
the carved well, and fill it with clean running buffer. (4) Continue to run the gel for 5 min while every 1 min collecting the
buffer from the well into a fresh tube and quickly replacing it
with fresh buffer. (5) Reimage the gel to validate that the
library band no longer appears and use TapeStation and a
D1000 ScreenTape to assess in which of the tubes the library
eluted, and to measure its concentration and average length.
5. Nascent RNAs incorporate 4SU during transcription; therefore
in most cases overcrowding of cells should be avoided to maintain a metabolically active culture. The 16-h labeling time
window can be increased for slow-growing cells, or decreased
for labeling of transient events, as required.
6. Fluorescence microscopy imaging should be performed
according to the standard procedures applied per lab, cells,
and experimental system. It is essential to image both the
APEX2-fusion protein and biotinylated proteins in all conditions (labeled and ÀBP, Àhydrogen peroxide, and ÀAPEX2
controls). Note that immunological detection of the APEX2fusion protein may vary after labeling due to self-biotinylation.
Biotinylated proteins can be labeled by fluorophore-coupled
neutravidin or an alternative (we use Alexa-Fluor-647-coupled
neutravidin). The main goals of imaging are (1) to confirm that
APEX2 is expressed and localized as expected; (2) to confirm
Proximity-CLIP
303