title: GSM4217923: ChIP WT LPS OBF1 rep1; Mus musculus; ChIP-Seq
library_construction_protocol: To extract the nuclei of stimulated splenic B cells: cultured CD19+ spleen B cells stimulated with either LPS or anti-CD40/IL4 were used for chromatin immunoprecipitation as described {Choukrallah, 2015 #46}. Immediately after centrifugation, supernatant was discarded, and cells were resuspended and fixed with 10 ml 1% formaldehyde in PBS at room temperature for 10min, followed by quenching with 530μl 2.5 M Glycine at room temperature for 5 min. Cells were lysed for 10 min in 10 ml lysis buffer A (10 mM HEPES, pH8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, PI). The supernatant was discarded, and pellets were incubated for 10 min in lysis buffer B (10 mM HEPES, pH8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100, PI). Then, the nuclei were resuspended in chromatin lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% SDS) for 30 min at 4 ℃. Subsequently, sonication was performed using Bioruptor Next Gen (Diagenode) at high output intensity for 25 cycles (30s on/30s off). Finally, the fragmented chromatin suspension was diluted 5 times with chromatin dilution buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 167 mM NaCl, 1.67% Triton X-100). 30-50 μg chromatin was subjected to immunoprecipitation with 5-10 μg ChIP-grade antibodies and incubated overnight. Subsequently, the samples were incubated with Dynabeads Protein G for 2 hours. The beads were washed with the following buffers: twice with low salt buffer (20 mM Tris pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), twice with high salt buffer (20 mM Tris pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), twice with LiCl buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% Na Deoxychholate) and once with TE (20 mM Tris-HCl, pH8.9, 2 mM EDTA). The protein-DNA complexes were eluted from the beads with 350 μl elution buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 1% SDS, 100 mM NaHCO3) and reverse crosslinked overnight at 65 ℃ with 60 μg Proteinase K and 60 μg RNase A. Immunoprecipitated DNA was recovered using AMPure XP beads. libraries were prepared using NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturers instructions
attributes.tag: GEO Accession
attributes.value: GSM4217923