12 mm round coverslips

Cells were cultured on 12 mm round coverslips (corning) and were fixed with 4% EM-grade paraformaldehyde (Electron Microscopy Science, Hatfield, PA) in PBS pH7.4 for 10 min at room temperature. Cells were then washed 3 x with PBS for 10 min each, treated with permeabilizing buffer (10% FBS in PBS) containing 0.1% saponin for 20 min and treated in blocking buffer for 30 min. Subsequently, cells were incubated with primary antibodies in permeabilizing buffer for 1 hr at room temperature, washed 3 x with PBS for 10 min each and incubated with secondary antibodies in permeabilizing buffer for 1 hr at room temperature and finally washed 3 x with PBS for 10 min each. Cells were mounted on slides with Prolong Gold with DAPI (Thermo Fisher Scientific, P36931). Primary antibodies used in the immunofluorescence studies were as follows: anti-YBX1 (Abcam, ab12148), YBAP1 (Santa Cruz Biotechnology, sc-271200). Images were acquired with Zeiss LSM900 confocal microscope and analyzed with the Fiji software (http://fiji.sc/Fiji).

The day prior to transfection, HEK293T cells were seeded at 70,000 cells per well in 24 well plates on 12 mm round coverslips (Corning). On the day of transfection, 300 ng of mRNA codifying for Cas9d-MG34-1 (3XFLAG) was complexed in Lipofectamine Messenger Max (Thermo Fischer Scientific) following the manufacturer’s instructions and the complex was added to cells. Twenty-four hours post-transfection, cells were fixed by adding 4% formaldehyde (Sigma Aldrich) for 20 min at room temperature. Subsequently, cells were washed three times with PBS and permeabilized/blocked by adding blocking buffer (0.1% TX-100 in 2% FBS) for 20 min. Cells were then incubated with 1:100 dilution of anti-FLAG (Sigma Aldrich, F3165), in blocking buffer for 1.5 h at room temperature, extensively washed and incubated in secondary antibodies diluted 1:1000 in blocking buffer and Alexa Fluor 488 (Thermo Fischer Scientific, A32723) for 1.5 h. Cells were extensively washed, rinsed briefly in dH₂O and mounted on slides with ProLong Gold with DAPI (Thermo Fischer Scientific). For antibody validation experiments, non-transfected HEK293T cells were imaged, keeping all the settings constant in an EVOS 5000 (Thermo Fisher Scientific).