RNA Detection

14. 1Hybridization buffer: 10 mM Tris–HCl pH 7.5, 5 mM
    EDTA, 500 mM LiCl, 0.5% DDM, 0.2% SDS, 0.1% sodium
    deoxycholate, 4 M urea, and 2.5 mM TCEP.


15. Total cell lysis buffer: 10 mM Tris–HCl pH 7.5, 500 mM LiCl,
    0.5% DDM, 0.2% SDS, 0.1% sodium deoxycholate, 1prote-
    ase inhibitor cocktail, and 1000 U/mL murine RNase
    inhibitor.


16. Benzonase elution buffer: 20 mM Tris–HCl pH 8.0, 0.05%
    NLS, 2 mM MgCl 2 , and 0.5 mM TCEP.


17. NLS elution buffer: 20 mM Tris–HCl pH 8.0, 10 mM EDTA,
    2% NLS, and 2.5 mM TCEP.


18. Bead based nucleic acid purification kit (We use Life Technol-
    ogies Dynabeads®MyOne™SILANE).

3 Methods

Maintain samples on ice and perform all centrifugation steps at 4C

unless otherwise noted. All buffers should be prepared before

starting the protocol. Buffers containing urea should be freshly

prepared, or alternatively, can be prepared then immediately ali-

quoted and frozen at 20 C. Do not store urea buffers at room

temperature for extended periods of time. Before beginning experi-

ments, select the target RNA of interest and controls as needed (see

Note 2) and determine number of input cells to be used for each

capture (seeNote 3). If the cellular localization of the RNA is

already known, select the appropriate lysis method for the target

RNA; otherwise, use the whole cell lysis method (seeNote 4).

3.1 Preparation of
SILAC Labeled Cell
Pellets

1. Initiate culture of cell line of interest. Once cells are growing
   well, split into two parallel cultures in SILAC Heavy and
   SILAC Light medium and grow for at least three passages to
   incorporate SILAC labels (seeNote 5).


2. Seed adherent cells on 15 cm tissue culture plate. Grow to
   70–90% confluence then remove medium from plate and
   replace with 10 mL ice-cold 1PBS. Rock gently for 10 s
   then remove PBS wash. Add another 10 mL ice-cold PBS to
   plate to prevent cells from drying during cross-linking.


3. Place plates on a shallow tray of ice and UV cross-link at
   254 nm for a total energy of 0.8 J/cm^2. Remove plates from
   cross-linker and keep on ice for the remainder of the procedure.


4. Scrape cells from the plate using a cell lifter and transfer to a
   sterile 15 mL tube. Cell suspensions from multiple plates can
   be pooled at this point.


5. Pellet cells by centrifugation at 1000gfor 5 min.

Identification of Direct RNA Binding Proteins Using RAP-MS 479