Epigenetic Profiling of Neonatal Immune Cells

ChIP-seq was performed on purified CD14+ monocytes from healthy and chorioamnionitis-exposed term infants, preterm infants, and extremely preterm infants as previously described[17 (link)]. Each age group contained 3 biological replicates. In order to obtain approximately 5×10⁵ cells per immunoprecipitation, each neonatal sample replicate contained 3 pooled samples (Table 11). The ultrasonication was performed on wet ice using the Branson Digital Sonifier 450 for 240 seconds at 40% amplitude with 0.7 seconds ‘on’ and 1.3 seconds ‘off’. The immunoprecipitation was performed with 4 micrograms of H3K4me3 antibody (Abcam, Cambridge, MA, USA, ab8580). After library preparation, the samples were pooled in equimolar concentrations and single-end ChIP-seq (50 nt) was performed on an Illumina HiSeq 2500 machine, resulting in 20–100 million reads/sample. The quality of the samples was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and ChIPQC[53 (link)]. All samples passed quality control measures, which are outlined in Table 12.