Sx 8g ip star robot

Methyl-CpG immunoprecipitation (MCIp) was performed as described previously [24 (link)]. In brief, a total of 2.5 μg DNA was sonicated with the Bioruptor NextGen (Diagenode, Liege, Belgium) to fragments of 100 to 600 bp as monitored on a 1.5% agarose gel. MCIp enrichment of highly methylated DNA was performed, as described, with minor modifications using SX-8G IP-Star robot (Diagenode). Sonicated DNA was enriched with 90 μg purified methyl-CpG-binding domain-Fc protein coupled to 60 μl protein A-coated magnetic beads (Diagenode). DNA was eluted by incubation with increasing NaCl concentrations (fraction A, 300 mM; B, 400 mM; C, 500 mM; D, 550 mM; E, 1,000 mM). Desalted eluates were controlled for enrichment of methylated DNA by real-time PCR analyzing the imprinted gene Mest. The non-methylated allele elutes at low-salt while the methylated allele elutes at high-salt concentration.

MCIp enrichment of highly methylated DNA and CpG island microarray analysis was performed as described previously with minor modifications [50 (link), 51 (link)]. Briefly, 2.5 µg DNA was sonicated with the Bioruptor NGS (Diagenode, Liege, Belgium) to fragments of 100 to 600 basepairs (bp). Fragmented DNA was MCIp-enriched using an SX-8G IP-Star robot (Diagenode) and 60 µg MBD2-Fc protein coupled to protein A magnetic beads (Diagenode). Proper DNA enrichment was monitored by quantitative real-time PCR targeting the imprinted gene SNRPN. The non-methylated allele elutes at low-salt, while the methylated allele elutes at high-salt concentration. Highly methylated tumor and healthy control DNA (matched normal mucosa from nearby the tumor) were labeled with Alexa Fluor 5 and 3, respectively, and cohybridized to a 244 K human CpG island microarray (Agilent, Germany, Böblingen) covering 27800 CpG islands represented by 199,399 probe sequences with a length of 45–60 bp per probe sequence.

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.