- 0.4 M Arg10 solution: Reconstitute 62.1 mg of^13 C 6 ,^15 N 4 L-
arginine HCl (Cambridge Isotope Laboratories) in 700μL
deionized water. Store at 20 C. - 0.8 M Lys8 solution. Reconstitute 128.1 mg of^13 C 6 ,^15 N 4 L-
lysine 2HCl (Cambridge Isotope Laboratories) in 700μL deio-
nized water. Store at 20 C. - SILAC “heavy” cell culture medium: Mix 0.5 L of high glucose
SILAC DMEM (PAA or Thermo Fisher Scientific), 50 mL of
dialyzed fetal bovine serum, 5 mL of 200 mML-glutamine,
5 mL of 10,000 U/mL penicillin-streptomycin, 0.5 mL of
0.4 M Arg10 and 0.5 mL of 0.8 M Lys8. Filter the medium
through 0.2μm filter membrane and store at 4C.
3 Methods
3.1 Cell Culture and
UV Cross-Linking
- Culture adherent cells in desired medium until 40–70 % con-
fluence and incubate them in the presence of 100–200μM
4-thiouridine (4SU) for 6–10 h. Duration and concentration
of 4SU treatment may have to be optimized (seeNote 1). - (Optional: Apply an additional labeling pulse with 100μM
4SU 1–2 h prior to UV cross-linking to ensure the labeling of
short-lived transcripts.) - Decant culture medium and place the dishes on ice. Wash once
with 5–10 mL ice-cold PBS to remove the residual culture
medium. Make sure you remove most of the liquid before
exposure to UV light (seeNote 2). Next, cross-link cells with
UV light with a wavelength of 365 nm and energy of 0.2 J/cm^2
using a UV cross-linker. Repeat this process for all remaining
dishes in culture. - Immediately harvest UV-exposed cells by scraping them off in
5 mL of ice-cold PBS using a cell scraper. Process a second
batch of cells without exposing them to UV light. This sample
serves as a background control. - Transfer cell suspensions into 50-mL falcon tubes and pellet
cells by centrifugation with 400gat 4C for 5 min. Wash the
cell pellet once with ice-cold PBS. Collect cells and freeze
pellets in liquid nitrogen or continue with the cell lysis.
3.2 Cell Lysis and
Oligo(dT) Purification
- Lyse cells in approximately five times the cell pellet volume of
lysis/binding buffer containing 5 mM DTT and Complete
Mini EDTA-free protease inhibitor cocktail by pipetting up
and down and incubate the extract for 15 min on ice (see
Note 3). If sample volumes exceed 50 mL, aliquot in several
50-mL falcon tubes.
Isolation and Differential Analysis of RBPs 409