GSM7430947: SM_NONO_Eto; Homo sapiens; OTHER
Cells cultured in the absence or presence of etoposide were washed with PBS, subjected to UV irradiation (200 mJ/cm2), scraped and resuspended in ice-cold PBS, pelleted (1200 rpm, 5 min) and stored at -80 °C. The pellet was lysed (20 min, 4°C) in eCLIP lysis buffer (50 mM tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.25 mM of TCEP) and sonicated. Subsequently, a limited RNase I (Invitrogen) digest (10 min, 22 °C) and TURBOTM DNase (Invitrogen) digest (20 min, 37 °C) was performed. For the immunoprecipitation (IP) per mg total protein, 5 µg of anti- NONO rabbit polyclonal antibody (proteintech®; cat: 11058-1-AP; lot: 0082415) was coupled to 30µl DynabeadsTM Protein G beads (Invitrogen) and incubated over night at 4 °C. The samples were washed twice in CLIP lysis buffer, twice in wash buffer (50 mM of tris-HCl pH7.4, 300 mM of NaCl, 1 mM of EDTA, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.25 mM of TCEP), followed by two washes in low-salt wash buffer (50 mM of tris-HCl pH7.4, 1 mM of EDTA, 0.5% IGEPAL CA-630). Subsequent library preparation was performed as described in the eCLIP protocol (Nostrand, E. L. V. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–14 (2016)