Illumina HiSeq 2500 sequencing; Cyclin D1 ChIP-seq in H9 undifferentiated human pluripotent stem cells
Cyclin D1 ChIP-seq in H9 undifferentiated human pluripotent stem cells
hESCs were washed with PBS and detached from the plate by incubating them for 10 min at 37 Â°C in Cell Dissociation Buffer (Gibco). ChIP was carried out as described before (Bienvenu et al. 2010; Casimiro et al. 2012; Pauklin and Vallier 2013), except that crosslinking was performed in solution in PBS if samples were sorted by FACS. Data for each cell cycle was normalized to IgG control of each cell cycle phase. We used additional controls as follows: 1) Input of each cell cycle phase and 2) primers for a chromatin region downstream of Smad7 locus known to be negative for histone marks. All of these controls supported our results indicating an enrichment of Cyclin D proteins to neuroectoderm and endoderm loci in late G1 phase. Antibodies for Cyclin D ChIP have been used previously (Landis et al. 2006). Two biological replicates and one input DNA as control sample were sequenced at the Cambridge Institute NGS service of the University of Cambridge using Illumina HiSeq. Nucleic acid libraries were constructed by the Cambridge SCI Sequencing facility
Experimental Factor: total genomic DNA
immunoprecipitate