21. 2Â Formamide gel-loading solution: 95% Formamide, 0.02%
(w/v) bromophenol blue, 0.01% (w/v) xylene cyanol,
10 mM EDTA.
22. Urea-PAGE (see Note 3).
23. Gel breaker tubes.
24. 5 μm Filter tube.
25. Pippin Prep and 3% Pippin gel cassettes (Sage Science) (see
Note 4).
26. DNA clean and concentrate kit.
27. Phenol:chloroform:isoamyl alcohol 25:24:1, saturated with
10 mM Tris, pH 8.0, 1 mM EDTA.
28. Stranded, random hexamer primed RNA Library Prep Kit
(we use NEBNext Ultra™ II directional RNA Library prep
kit for Illumina).
29. Ribosomal RNA depletion kit (we use NEBNext rRNA depletion kit).
30. Magnetic rack for 96-well plate.
31. Agilent TapeStation with DNA D1000 and RNA ScreenTapes,
or Bioanalyzer (less recommended).
3 Methods
3.1 Setting Up
and Procedures Before
Proximity Labeling
and UV Cross-Linking
1. Accurately count and split cells ~36 h prior to proximity labeling and cross-linking. All cells should be in a similar growth
state and confluence at the day of experiment. Balance the need
for cell number with the requirement of cells to be rapidly
growing for efficient 4SU incorporation (see Note 1 and
Fig. 2).
2. 16 h Before cross-linking, add 4-thiouridine (4SU) to the cell
culture media at a final concentration of 100 μM. The stock
concentration might be too high; calculate the required
amount and prepare an intermediate 50 mM 4SU solution.
Then add at 2 μL per ml of medium. It is not necessary to add
4SU to the media of cells grown for immunofluorescence
validations. Read more on 4SU labeling in the Notes section
(see Note 5).
3. Prepare stock solutions and buffers as described in Subheading
2.2.
3.2 Proximity
Biotinylation and UV
Cross-Linking
1. Bring cell media to 37
C and PBS to room temperature, label
1.5 mL tubes and pre-chill them on ice, have liquid N 2 on hand
for snap freezing cell samples, and an accessible sonicator
(in case Trolox does not dissolve). Weigh (but do not dissolve)
required amounts of sodium ascorbate and Trolox.
288
Daniel Benhalevy and Markus Hafner
(w/v) bromophenol blue, 0.01% (w/v) xylene cyanol,
10 mM EDTA.
22. Urea-PAGE (see Note 3).
23. Gel breaker tubes.
24. 5 μm Filter tube.
25. Pippin Prep and 3% Pippin gel cassettes (Sage Science) (see
Note 4).
26. DNA clean and concentrate kit.
27. Phenol:chloroform:isoamyl alcohol 25:24:1, saturated with
10 mM Tris, pH 8.0, 1 mM EDTA.
28. Stranded, random hexamer primed RNA Library Prep Kit
(we use NEBNext Ultra™ II directional RNA Library prep
kit for Illumina).
29. Ribosomal RNA depletion kit (we use NEBNext rRNA depletion kit).
30. Magnetic rack for 96-well plate.
31. Agilent TapeStation with DNA D1000 and RNA ScreenTapes,
or Bioanalyzer (less recommended).
3 Methods
3.1 Setting Up
and Procedures Before
Proximity Labeling
and UV Cross-Linking
1. Accurately count and split cells ~36 h prior to proximity labeling and cross-linking. All cells should be in a similar growth
state and confluence at the day of experiment. Balance the need
for cell number with the requirement of cells to be rapidly
growing for efficient 4SU incorporation (see Note 1 and
Fig. 2).
2. 16 h Before cross-linking, add 4-thiouridine (4SU) to the cell
culture media at a final concentration of 100 μM. The stock
concentration might be too high; calculate the required
amount and prepare an intermediate 50 mM 4SU solution.
Then add at 2 μL per ml of medium. It is not necessary to add
4SU to the media of cells grown for immunofluorescence
validations. Read more on 4SU labeling in the Notes section
(see Note 5).
3. Prepare stock solutions and buffers as described in Subheading
2.2.
3.2 Proximity
Biotinylation and UV
Cross-Linking
1. Bring cell media to 37
C and PBS to room temperature, label
1.5 mL tubes and pre-chill them on ice, have liquid N 2 on hand
for snap freezing cell samples, and an accessible sonicator
(in case Trolox does not dissolve). Weigh (but do not dissolve)
required amounts of sodium ascorbate and Trolox.
288
Daniel Benhalevy and Markus Hafner