Pooled genomic DNA from lungs of control animals
0.5 ug of pooled genomic DNA was digested with 400U MspI (NEB) in 100ul at 37C for 18-24 hours followed by QIAQuick purification (Qiagen, Valencia, CA). The end of digested DNA was filled in and then an “A” base was added to the 3’ end of the DNA fragments according to Illumina standard end repair and add_A protocol (Illumina, San Diego, CA). Pre-annealed forked Illumina adaptor containing 5’-methyl-cytosine instead of cytosine was ligated to both ends of DNA fragments using standard Illumina adaptor ligation protocol (Illumina, San Diego, CA). Ligated fragments were then separated by 2% agarose gel (Sigma). Two size ranges 150-175bp and 175-225bp (size including adaptor length) were selected from the gel. DNA from gel slices were purified using Qiagen Gel extraction kit (Qiagen Valencia, CA). Purified DNA were subsequently treated with EpiTect Bisulfite kit (Qiagen, Valencia, CA) with the following modification: the bisulfite conversion time was extended to approximately 14 hours by adding 3 cycles of 95C 5 minutes denaturation followed by 60C 180 minutes incubation. Then bisulfite converted DNA was purified following purification protocol for DNA isolated from FFPE tissue sample in the EpiTect Bisulfite kit, except the following step. The desulfonation buffer was added to the spin column and incubated for 20 minutes instead of 15 minutes at room temperature. Then bisulfite treated DNA was purified one more time with MinElute PCR purification kit (Qiagen, Valencia, CA) and elute into 15 ul EB buffer. Bisulfite treated DNA fragments were enriched in the following PCR reaction: 15 ul of eluted DNA, 5 pmol of Illumina PE PCR primers 1.0 and 2.0, 62.5nM of each dNTPs and 2.5 U of Pfu Turbo Cx hotstart DNA polymerase (Stratagene) in a total 50ul volume, 5 min at 95C, 30 sec at 98C then 6X (10 sec at 98C, 30 sec at 65C, 30 sec at 72C) followed by 5 min at 72C. PCR reaction was purified by MinElute PCR purification Kit (Qiagen, Valencia, CA) and final RRBS library was eluted in 15 ul EB. The concentration of final library was determined by the Agilent 2100 Bioanalyzer (Palo Alto, CA) and cluster was generated and sequenced on Illumina GA sequencing machine according to standard Illumina cluster generation and sequencing protocols.