4. Wash streptavidin-coupled magnetic beads twice with RIPA
buffer. Then incubate with rotation 1.5 mg total protein of
the cell extracts with ~60 μL of washed beads (see Note 7) for
1 h at room temperature. Keep 30 μL of the extract sample for
total RNA extraction and store the rest for Western blot
analyses.
5. Collect the beads using a magnetic rack and transfer the supernatant into pre-labeled and chilled tubes.
6. Wash beads with 1 mL volume of the following ice-cold solutions: twice with RIPA buffer, once with 1 M KCl, once with
0.1 M Na 2 CO 3 , once with 2 M urea in 10 mM Tris–HCl pH 8
(freshly made), and again twice with RNase T1 buffer (see
Subheading 2.4).
7. Use the last wash to split the beads from each cell sample to
three non-equal aliquots: 30% for mass spectrometric analysis.
Reduce liquid volume to minimum and keep on ice. 20% for
RNA-seq of intact bound RNAs; collect beads on a magnetic
rack, discard supernatant, and store at À80
C (note that this
will spoil the magnetic beads). Keep the remaining 50% of the
beads for RNA footprinting and small RNA cDNA library
preparation. At this point it is necessary to continue with the
beads for RNA footprints and proteomic analysis. We recommend proceeding first with Subheading 3.6, steps 1–12, and
then returning to perform Subheading 3.5, steps 1–5, before
pausing.
ä
Fig. 3 (continued) Western blot analysis as in a of the eluted material (Subheading 3.3, step 7), using
decreasing volumes of beads per identical volume of extract. (d) Western blot analysis as in c of the unbound
material with different volumes of beads used (Subheading 3.3, step 5), relative to the input (cell extract). (e)
Immunofluorescence analysis of cells expressing a CNX43-EGFP-APEX2 fusion protein. Note that while
biotinylation (in yellow, detected by fluorophore-coupled neutravidin) is concentrated as expected at cellcell interfaces, the GFP signal is also highly detected in cytoplasmic foci. (f) Anti-GFP Western blot analysis of
cells as in e, illustrating that while the intact fusion protein is expressed, additional shorter product suggests
proteolytic cleavage. The relative specificity of biotinylation to cell-cell interface suggests that the majority of
cleavage events result in nonfunctional APEX2, while a free EGFP product remains stable. (g) TapeStation
analysis of the intact RNA (not RNase treated) eluted from beads (Subheading 3.7, step 9) after Proximity-CLIP
in HEK293 T-Rex cells that do not express APEX2 and of cells that express either NES and CNX43 APEX2fusion proteins. Signal over 25 nt length represents the internal control of the TapeStation analysis. It is
essential to sequence the RNA eluted from negative control cells even though quantitatively the amount of
eluted RNA appears lower
Proximity-CLIP
293
buffer. Then incubate with rotation 1.5 mg total protein of
the cell extracts with ~60 μL of washed beads (see Note 7) for
1 h at room temperature. Keep 30 μL of the extract sample for
total RNA extraction and store the rest for Western blot
analyses.
5. Collect the beads using a magnetic rack and transfer the supernatant into pre-labeled and chilled tubes.
6. Wash beads with 1 mL volume of the following ice-cold solutions: twice with RIPA buffer, once with 1 M KCl, once with
0.1 M Na 2 CO 3 , once with 2 M urea in 10 mM Tris–HCl pH 8
(freshly made), and again twice with RNase T1 buffer (see
Subheading 2.4).
7. Use the last wash to split the beads from each cell sample to
three non-equal aliquots: 30% for mass spectrometric analysis.
Reduce liquid volume to minimum and keep on ice. 20% for
RNA-seq of intact bound RNAs; collect beads on a magnetic
rack, discard supernatant, and store at À80
C (note that this
will spoil the magnetic beads). Keep the remaining 50% of the
beads for RNA footprinting and small RNA cDNA library
preparation. At this point it is necessary to continue with the
beads for RNA footprints and proteomic analysis. We recommend proceeding first with Subheading 3.6, steps 1–12, and
then returning to perform Subheading 3.5, steps 1–5, before
pausing.
ä
Fig. 3 (continued) Western blot analysis as in a of the eluted material (Subheading 3.3, step 7), using
decreasing volumes of beads per identical volume of extract. (d) Western blot analysis as in c of the unbound
material with different volumes of beads used (Subheading 3.3, step 5), relative to the input (cell extract). (e)
Immunofluorescence analysis of cells expressing a CNX43-EGFP-APEX2 fusion protein. Note that while
biotinylation (in yellow, detected by fluorophore-coupled neutravidin) is concentrated as expected at cellcell interfaces, the GFP signal is also highly detected in cytoplasmic foci. (f) Anti-GFP Western blot analysis of
cells as in e, illustrating that while the intact fusion protein is expressed, additional shorter product suggests
proteolytic cleavage. The relative specificity of biotinylation to cell-cell interface suggests that the majority of
cleavage events result in nonfunctional APEX2, while a free EGFP product remains stable. (g) TapeStation
analysis of the intact RNA (not RNase treated) eluted from beads (Subheading 3.7, step 9) after Proximity-CLIP
in HEK293 T-Rex cells that do not express APEX2 and of cells that express either NES and CNX43 APEX2fusion proteins. Signal over 25 nt length represents the internal control of the TapeStation analysis. It is
essential to sequence the RNA eluted from negative control cells even though quantitatively the amount of
eluted RNA appears lower
Proximity-CLIP
293