GSM5320877: Input DNA ESC Even set; Mus musculus; OTHER
ChIRP experiments were performed using the described protocol. A total of 48 antisense oligonucleotide biotinylated probes against lncSmad7 were designed with Stellaris Probe Designer and were split for the ChIRP experiments in two subsets (odd and even). LacZ-specific probes were used as negative controls. Briefly, 5×107 lncSmad7-overexpressing ESCs were cross-linked with 1% formaldehyde for 10 min at RT and processed to obtain nuclear extracts as described in Native RIP protocol. The isolated nuclei pellets were lysed with 1% SDS Lysis buffer (50 mM Tris pH 7.0, 10 mM EDTA, 1% SDS, 1 mM DTT, protease inhibitors). The lysate was sonicated using the Bioruptor Twin (Diagenode) (20 cycles, 30s on/off) and incubated with biotinylated probes in hybridization buffer (500 mM NaCl, 1% SDS, 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 15% formamide, protease inhibitors cocktail, PMSF, and RNase inhibitor) for 4 hours at 37°C with shaking. After hybridization and washes with ChIRP wash buffer (2xSSC, 0.5% SDS, 1 mM DTT, protease inhibitors), the complexes were recovered by incubation with MyOne Streptavidin C1 Dynabeads (Invitrogen) in a total volume of 1 ml and associated DNA fragments from 900 ul of bead samples were purified using acidic phenol/chloroform (ThermoFisher) and ethanol precipitated. For sequencing of ChIRP samples, approximately 10 ng of purified ChIRP DNA were end-repaired, dA-tailed, and adaptor-ligated using the NEBNext ChIP-seq Library Prep Master Mix Set (NEB), according to the manufacturer's instructions. In parallel, from 100 ul of bead samples, RNA was isolated in a buffer with 95% formamide, 10 mM EDTA pH 8 for 10 min at 90°C followed by TRIzol extraction (Invitrogen). ChIRP libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB), according to manufacturer's instructions and were checked by Bioanalyzer (Agilent) for quality.