ChIP-seq and RNA-seq of Endothelial Cells

Formaldehyde cross-linked chromatin was sonicated using Covaris S220 to 200–300 bp and ChIP was performed using SX-8G IP-STAR robot (Diagenode) using the AF4798 antibody. ChIP-Seq library was prepared using ChIPmentation procedure and libraries were sequenced using Illumina HiSeq. 2500 at CCHMC sequencing core. Data analysis was performed using the BioWardrobe platform^{29 (link)}. Briefly, ChIP-Seq reads were aligned by Bowtie to the mouse genome (mm10); only unique reads with no more than one mismatch were kept. Reads were extended to estimated fragment length, normalized to total mapped read number and displayed as coverage on a mirror of the University of California Santa Cruz (UCSC) genome browser. MACS2^{30 (link)} was used to identify islands of enrichment. FOXF1 sequence logos were identified with MEME-ChIP^{31 (link)}. FOXF1 ChIPseq data was aligned with previously published ChIPseq data for histone methylation (GEO Accession GSE31039) using the BioWardrobe platform.
RNAseq analysis was performed on FACS-sorted endothelial cells (CD45⁻CD31⁺CD326⁻) from control and PDGFb-iCre/Foxf1^fl/+ lungs 4 days after PNX surgery. Changes in gene expression were determined using DESeq and analyzed as previously described^{11 (link)}. Heat map was generated from differentially expressed genes using JMP Genomics 6.0.
ChIPseq and RNAseq data are available as GEO accession GSE100149.