title: GSM4763681: Ebp1_IP_Rep1; Mus musculus; RIP-Seq
library_construction_protocol: Cells were washed and detached in 10 mL ice-cold PBS supplemented with 12 mM MgCl2 and 100 µg/mL cycloheximide (Sigma-Aldrich, C7698) and centrifuged for 4 min at 300 xg, 4 °C. The cell pellet was lysed in 0.5 mL lysis buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 12 mM MgCl2, 5mM CaCl2, 0.5 % NP-40, 100 µg/mL cycloheximide, 25 U/mL DNase-I (Roche, 4716728001), 1x Protease Inhibitor cOmplete EDTA-free (Roche, 05056489001), 1 mM PMSF (Roth, 6367)). The lysate was passed five times through a 23 G needle and cleared by centrifugation (2 min at 13000 xg, 4 °C). After clearing the lysate by centrifugation (2 min at 13000 xg, 4 °C), the supernatant was divided for total (200 µL) and IP (400 µL) translatome samples. Total samples were digested with MNase (300 U / 1 A260 nm) for 30 min at 4 °C, under rotation. The reaction was terminated by addition of 10 mM EGTA and ribosomes were purified by sucrose cushion centrifugation (25 % sucrose, 20 mM Tris-HCl pH 8.0, 140 mM KCl, 12 mM MgCl2, 100 µg/mL cycloheximide, 1 mM PMSF) for 90 min, 75000 rpm, at 4 °C (Beckmann AT2 S120 rotor). Pelleted ribosomes were resuspended in lysis buffer lacking CaCl2 and subjected to phenol-chloroform extraction of RNA. IP samples were mixed with 100 µL Dynabeads (Life Technologies, 10008D) conjugated with 10 µg anti-Ebp1CT (Abcam, ab35424) and digested using MNase (300U / 1 A260nm) for 30 min at 4 °C, under rotation. The reaction was terminated by addition of 10mM EGTA and the beads were washed three times in 400 µL wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 12mM MgCl2, 1 mM PMSF, 100 µg/mL cycloheximide, 0.1 % NP-40) followed by phenol-chloroform extraction of RNA. Gel purification and 3'-dephosphorylation of ribosome-protected footprints from total and IP samples was performed as previously described (Galmozzi et al., 2019). Footprints were then 5'-phosphorylated using T4 PNK (NEB, M0201S) and ATP for 1 hr at 37 °C. Deep sequencing libraries were prepared from these RNA fragments using the NEXTflex Small RNA-seq Kit v3 (Bio Scientific, NOVA-5132-06).
attributes.tag: GEO Accession
attributes.value: GSM4763681