GSM8155085: hPSC Pancreatic Endocrine Progenitor Monolayer, NEUROG3 AF3444, ChIP, Rep 2, Batch 2; Homo sapiens; ChIP-Seq
Collected samples were cross-linked in 1% formaldehyde in PBS for 12 min at room temperature and was stopped by replacing with 0.125 M glycine. Nuclei was pelleted in lysis buffer. The desired amount of fragmented chromatin was supplemented with Nuclear Lysis Dilution Buffer and precleared with blocked Protein G magnetic beads (Thermo Fisher Scientific) with rotation at 4 oC for 3 h. 1% precleared samples were saved as input and each 25μg sample was incubated with 20μg Protein G magnetic beads preloaded with 5ug antibody overnight with rotation at 4 oC. Beads were then washed sequentially at 4 oC. Protein-DNA complexes were eluted from the beads in Elution Buffer (50 mM Tris-HCl pH=8, 10 mM EDTA, 1% SDS) at 65°C for 30min. Cross-links were reversed at 65°C overnight, and DNA was purified with phenol:chloroform, chloroform and ethanol for library construction ChIP-seq libraries was prepared and sequenced at CCHMC DNA sequencing and Genotyping Core Facility. DNA libraries were prepared using 5-10 ng of starting material using the SMARTer ThruPLEX DNA-Seq kit (Takara; R400674). After library amplification, DNA was purified using AMPure XP beads (Beckman-Coulter; A63880) and size-selected. DNA fragment traces were analyzed on a Bioanalyzer.