Supplemental Methods Proband and Control Lung Samples Lung Sections from Both the Proband and Age-Matched Controls Were Obtained

Supplementary material J Med Genet

Supplemental Methods

Proband and Control Lung Samples

Lung sections from both the proband and age-matched controls were obtained in the form of fixed formalin paraffin embedded (FFPE) samples. Control lung samples were obtained from the BRINDL biorepository at the University of Rochester(1).

RNA expression analyses

RNA was obtained for mRNA expression studies using the RNeasy FFPE kit (Qiagen) according to manufacturer’s instructions. cDNA was then generated using iScrpit kit (biorad), and quantitative PCR was done as previously published (2). Primers for qPCR are listed in supplemental table 1.

Chromatin Immunoprecipitation of FFPE for Fixed Formalin Paraffin Embedded samples

Chromatin immunoprecipitation was done on FFPE sections using a protocol modified from Fanelli and colleagues, Nature Methods, 2011(3) Samples were deparaffinized by incubating 10uM sections in 1ml of Xylene for 10 minutes at room temperature. The tissue was then pelleted at 17,000 x gravity for 3 min at room temperature. This process was repeated 3 times. Samples were then rehydrated by incubating with 1 ml of 100% Ethanol for 10 minutes at RT. Cells were pelleted and resuspended in progressively increasing percentage of water as follows: 95% ethanol, 70% ethanol, 50% ethanol, 20% ethanol. The sample was then resuspended in 1x PBS and the tissue dissociated by sonicating with a Bioruptor (Diagnode, NJ USA) for 30 seconds on the medium setting. Cells then resuspended in Collagenase digestion buffer and treated with collagenase A (Roche 11 088 785 103) to a final concentration of 2mg/ml and digested at 37 C for 45 minutes. Cells were then pelleted and resuspended in ice-cold cell lysis buffer (10 m Tris, pH 8.0, 10 mM NaCl, 0.2% NP40, 5 mM sodium butyrate) and lysed on ice for 20 minutes. Cells were pelleted at 5000x g for 5 min and washed several times with cold PBS. The pellet was then re-suspended in room temperature Nuclei Lysis buffer (50 mM Tris, pH 8.0, 10mM EDTA, 1% SDS) and the nucei lysed on ice x 20 minutes. The lysate was diluted with 1x ChIP dilution buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS) and sonicated on Hi for 35 cycles of 30 seconds on/30 seconds off in the Diagenode Bioruptor. Samples were spun at max speed and supernatant moved to clean tube. The remaining ChIP protocol was completed as published (4), using an antibody against H3K27Ac (Abcam).

Array Comparative Genomic Hybridization Analysis

Array Comparative Genomic Hybridization (aCGH) Analysis was done using DNA from peripheral blood, extracted using QIAamp® DNA Blood Mini Kit (Cat #51106). Nanodrop ND-2000 spectrometer (Thermo Scientific, DE, USA) was used for determination of DNA concentrations. Microarray experiments were performed on SurePrint G3 ISCA CGH+SNP Microarray Kit, 4x180K v2.0 platform (Agilent Technologies, Santa Clara, CA), featuring approximately 110,715 custom