title: GSM3074865: Input_NEUROG2_U251_NSFD_1DPT; Homo sapiens; ChIP-Seq
library_construction_protocol: For chromatin immunoprecipitation experiment, U251 cell was cultured in 10 cm plate. After infected U251 cell with different prepared lentivirus, U251 cell was treated with neuron induction medium supplemented with forskolin and dorsomorphin as same as glioblastoma reprogramming process. Approximately total ~16×106 cells (4 10cm plates) were crosslinked with methanol-free formaldehyde (Thermo Scientific, 28908) at final concentration of 1% v/v for 8 minutes. Then quench the crosslinking with glycine at final concentration of 0.125 M for 5 minutes. Place plates contain U251 cells on ice, quickly twice washed with 10 ml pre-cold PBS, scraped and collected cell by centrifugation (525g, 5 minutes, 4°C). Aspirated the supernatant and resuspended cell pellet in 5 ml lysis buffer ((100 mM HEPES (pH 8.0), 85 mM KCl, 1% (v/v) IGEPAL CA-630, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail (Roche, 11873580001))) and incubated on ice for 20 minutes. Dounce homogenized U251 cells (20 repetitions) on ice and performed centrifugation (525g, 5 minutes, 4°C) to collected cell nuclei. Resuspended cell nuclei pellet in shearing buffer (50 mM HEPES (pH 8.0), 10 mM EDTA (pH 8.0), 1% (w/v) SDS, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail) and transferred to a TPX microtube (Diagenode, C-30010010-50) on ice. Shearing cell chromatin using a Bioruptor machine (Diagenode, B01010002). After Shearing, the sheared chromatin was centrifuged at 21,130 × g speed for 10 minutes at 4°C. Supernatant was collected and chromatin concentration was determined. Two samples of 10 ug chromatin has been saved, one was for input, the other was used for check chromatin shearing quality. 150 ug of chromatin with five-fold diluted by IP buffer (50 mM HEPES (pH 8.0), 20 mM NaCl, 1 mM EDTA (pH 8.0), 0.1% (v/v) Triton X-100, and 1% (v/v) cOmplete EDTA-free Protease Inhibitor Cocktail) was used for performing immunoprecipitation. HA-NGN2 or HA-SOX4/11 bound DNA fragments were immunoprecipitated with 5ug HA antibody (Abcam, AB9110). Histone H3 acetyl K27 bound DNA fragments were immunoprecipitated with histone H3 acetyl K27 antibody (Abcam, ab4729). Immunoprecipitation samples were incubated for 18 hours at 4°C with gentle rotation. Subsequently, 100 µl Magnetic Protein G Dynabeads (Life Technologies, 10003D) were resuspended in immunoprecipitation samples incubate for 2 hours at 4°C with gentle rotation to combine HA antibody. After magnetically isolation and washed, chromatin was eluted by adding 100 µl elution buffer (1% (w/v) SDS and 50 mM NaHCO3). The 10 ug chromatin input sample was eluted by 85ul elution buffer. Place those Input and IP sample to vigorous agitation (1,400 rpm, 27 °C, 30 minutes, Eppendorf Thermomixer). After subsequently treated with RNase, Proteinase K and NaCl(5M), Input and IP Chromatin was purified with a QIAquick PCR purification kit (Qiagen, 28104) according to kit manual. The chromatin concentration was determined by Qubit fluorometer and Qubit dsDNA HS assay kit (Life Technologies, Q32850) according to kit manual. 10 ng of input chromatin and 10 of HA-NGN2 or HA-SOX4/11 immunoprecipitated chromatin samples were used to build libraries by NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina kit (New England Biolabs, E6240S) for DNA sequencing. Duplicate or replicate libraries were built for each group from independent immunoprecipitations. After check library quality by bioanalyzer, DNA samples were submitted to sequencing core. Single end 50 base sequencing reads were generated and aligned to GRCh37/hg19. HOMER was used to the following analysis including Peak calling, annotation, motif discovery and GO analysis. Also, the UCSC Genome Browser was used to visualization of DNA sequencing data. Libraries were synthesized from 10 ng purified input chromatin and 10 ng purified immunoprecipitated chromatin using a NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs, E6240S) with NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7335S). Replicate libraries were prepared from independent immunoprecipitations.
attributes.tag: GEO Accession
attributes.value: GSM3074865