Mondrian sp workstation system

Chromatin immunoprecipitation was performed as previously described (Mousavi et al., 2012 (link)) using antibodies against Spt6 (Novus), KDM6A (Sigma), Suz12 (Cell signaling), Ezh2 (Cell signaling), H3K27me3 (Cell signaling), H3K27ac (Abcam), H3K36me3 (Abcam), Gal4 (Millipore), Flag (Sigma, F3165), Myc (Sigma), IgG (Abcam). Real-time PCR was performed with a SyberGreen MasterMix (Applied Biosystems) on a StepOnePlus realtime PCR system (Applied Biosystems). Oligonucleotides employed in ChIP-qPCR for the Gal4-TK-luc system are reported in Supplemental Information. For sequencing, the DNA fragments were blunt-end ligated to the Illumina adaptors and amplified via Mondrian SP Workstation system (NuGEN). Libraries were sequenced for 50 cycles on Illumina HiSeq 2000, HiSeq 2500 or HiSeq3000. Mock DNA (input DNA) was used against the matched sample data to call enriched regions and control for the false-positive detection rate (FDR). ChIP-seq data of H3K27me3, H3K27ac, H3K4me1, H3K4me3 in mouse ESCs were published in (Juan et al., 2016 (link)). ChIP-seq data of Med1, Oct4, Sox2, and Nanog in mouse ESC (Whyte et al., 2013 (link)) were downloaded and processed with the same settings.

Chromatin immunoprecipitation was performed as previously described (Mousavi et al., 2012 (link)) using antibodies against SMC3 (Abcam, Ab9236), NIPBL (Bethyl, A301-779A), RNA polymerase II (Abcam) or IgG (Abcam). Real-time PCR was performed with a SyberGreen MasterMix (Applied Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). Oligonucleotides employed in ChIP-qPCR for the are reported in Supplemental Information. For sequencing, the DNA fragments were blunt-end ligated to the Illumina adaptors and amplified via Mondrian SP Workstation system (NuGEN). Libraries were sequenced for 50 cycles on Illumina HiSeq3000. Mock DNA (input DNA) was used against the matched sample data to call enriched regions and control for the false-positive detection rate (FDR).