GSM5179354: KO rep1; Mus musculus; ChIP-Seq
For each experiment, single-cell suspensions from E12.5 neurospheres (passage 3) were collected. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched with 0.125 M glycine for 5 min. Cross-linked samples were then rinsed in PBS twice, and harvested in ice-cold IP buffer [100 mM NaCl, 50 mM tris-HCl (pH 8.1), 5 mM EDTA (pH 8.0), 0.02% NaN3, 0.5% SDS, 1× protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride], followed by sonication in a Bioruptor Pico (Diagenode) at a setting of “30 s on/30 s off, 30 cycles” at 4°C. Twenty microliters of aliquot was taken for checking the efficiency of sonication. Thirty microliters of lysate (3%) was kept to quantify DNA before immunoprecipitation (input). Immunoprecipitation was performed overnight at 4°C on a rotating wheel with sheared chromatin and indicated antibodies: 3 μg of rabbit anti-SMARCA4/BRG1 antibody (Proteintech, 21634-1-AP), and 3 μg of control IgG antibody (ABclonal, AC005). The next day, immunocomplexes were incubated with 50 μl of Protein G Sepharose beads for 4 hours at 4°C, followed by washing three times with wash buffer I [20 mM tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS] and once with wash buffer II [20 mM tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS]. Protein-DNA complexes were decross-linked in 120 μl of elution buffer (1% SDS and 0.1 M sodium bicarbonate) by shaking at 65°C for 3 hours. The elution was incubated overnight at 65°C with 2 μl of proteinase K (20 mg/ml) (Sangon Biotech) and 2 μl of RNase A (10 mg/ml) (TAKARA). DNA extraction, precipitation, and resuspension were performed using a DNA purification kit (QIAGEN). ChIP-seq libraries were constructed by StepWise DNA Lib Prep Kit for Illumina (Abclonal RK20202). For each sample, 40 μl purified ChIP DNA (about 300 pg) was end-repaired for dA tailing, followed by adaptor ligation with Full DNA Adapter Kit for Illumina Set_A (Abclonal RK20282). Each adaptor was marked with an index of 6 bp, which can be recognized after mixing different samples together. Adaptor-ligated ChIP DNA was purified by AMPure XP beads (1:1) and then used as template for 16 cycles of PCR amplification. Amplified ChIP DNA was purified again using AMPure XP beads (1:1) in 22 μl low-EDTA TE buffer. For multiplex sequencing, libraries with different index were mixed together with equal molar quantities by considering appropriate sequencing depth (20 million reads per library). Libraries were sequenced by Illumina Hi-seq X Ten platform with pair-end reads of 150 bp.
GEO
GSM5179354