GSM4083379: cKO input; Mus musculus; ChIP-Seq
For each experiment, single-cell suspensions from E13.5 dorsal cortices were collected as described above. Cells were cross-linked for 10 min at room temperature with 1% formaldehyde and then quenched with 0.125 M of 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× Roche protease inhibitor cocktail, and 1 mM PMSF), followed by sonication in a Bioruptor Pico (Diagenode) at a setting of '30s on/ 30s off, 30 cycles' at 4°C. 20 μl aliquot were taken for checking the efficiency of sonication. After de-crosslinked in de-crosslink buffer (1% SDS, 250mM NaCl in TE buffer) for 3 h, the sample was purified with DNA purification kit (QIAGEN), followed by DNA electrophoresis with 2% agarose gel. 30 μl lysate (3%) was kept to quantify DNA before immunoprecipitation (input). Immunoprecipitation was further performed with sheared chromatin and 3 μg anti-H3K36me3 antibody (abcam, ab9050) overnight at 4°C on a rotating wheel. Next day, immunocomplexes were incubated with 50 μl Protein G sepharose beads for 4 h at 4°C, followed by washing 3 times with wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 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, 0.1% SDS). Protein-DNA complexes were de-crosslinked in 120 μl de-crossed buffer (TE buffer) and shaked at 65℃ for 3 h. DNA extraction, precipitation, and resuspension were performed using 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.