Around 16 million of FACS purified neurons at E15.5 were collected, fixed and fragmented as described in ChIP-seq. The sequential ChIP experiment was performed using a Re-ChIP-IT kit (Active Motif). Anti-Pol II-Ser5P (ab5131, Abcam) or anti-H3K27me3 (07-449, EMD Millipore) was used in the first immunoprecipitation, respectively. Then anti-H3K27me3 or Anti-Pol II-Ser5P was added to precipitated chromatin in the second reaction. For control, a ChIP grade IgG (Abcam) was used replacing Anti-Pol II-Ser5P or anti-H3K27me3 in the second reactions. DNAs eluted from the second immunoprecipitations were applied to real-time quantitative PCR analysis using a DyNAmo flash SYBR green qPCR kit (Thermo Fisher) on a StepOnePlus real-time PCR system (Applied Biosystems).
Liu J., Wu X., Zhang H., Pfeifer G.P, & Lu Q. (2017). Dynamics of RNA polymerase II pausing and bivalent histone H3 methylation during neuronal differentiation in brain development. Cell reports, 20(6), 1307-1318.
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