GSM5643033: ChIRP_lncSMAD7-KO_EVEN_Rep2; Mus musculus; OTHER
ChIRP experiments were performed using the described protocol (Chu et al., 2011) in two replicates. 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). Briefly, 5×107 lncSmad7-overexpressing ESCs and lncSmad7 KO cells were cross-linked with 1% formaldehyde for 10 min at RT and processed to obtain nuclear extracts. 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 eluted with a cocktail of RNase A (Sigma-Aldrich) and RNase H (Invitrogen) at 37°C in DNA elution buffer (50 mM NaHCO3, 1% SDS, 200 mM NaCl) followed by reverse crosslinked at 65°C overnight, acidic phenol/chloroform (ThermoFisher) extraction and ethanol precipitated. Eluted DNA was subject to qPCR and high-throughput sequencing. In parallel, RNA was isolated in a buffer with 95% formamide and 10 mM EDTA pH 8.0 for 10 min at 90°C followed by TRIzol extraction (Invitrogen), from 100 ul of bead samples. Eluted RNA was subject to RT-qPCR analysis as reported for the detection of enriched transcript (Table 4). 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. ChIRP-seq
GEO Accession
GSM5643033