Control mice not exposed to arsenate
Mus musculus
Forty five day-old female C57BL/6 mice were obtained from Charles River Laboratories (Raleigh, NC) and acclimated for 2 weeks. The control mice were given distilled deionized water only. At the end of the study, mice were killed by exsanguination under CO2 anesthesia. The lung tissue was harvested immediately, placed in RNAlater™ (Ambion, Austin, TX), and stored at -70 °C.  A portion of frozen lung tissue was removed from each sample and used to isolate genomic DNA. Tissue from each treatment group was suspended in 1.2 ml cell lysis buffer (10 mM NaCl, 10 mM TrisCl pH 7.8, 2 mM EDTA) on ice and homogenized for 2 min (sterile 3 mm steel ball, Mixer Mill 301, Retsch, Newtown, PA). After addition of 125 µl 10% SDS solution and 23 µl Proteinase K (20 mg/ml) (Invitrogen, Carlsbad, CA) the homogenate was incubated at 55 °C for 3 h which was followed by the addition of 13 µl DNAse-free RNAse A (2 mg/ml) (Sigma-Aldrich, St. Louis, MO) and an incubation for 30 min at 37 °C. Protein precipitation was carried out by mixing 1340 µl tissue lysate 1:1 with Phenol:Chloroform:Isoamylalcohol (Invitrogen, Carlsbad, CA) in Phase Lock Gel Heavy Tubes (5Prime, Gaithersburg, MD) followed by 15 min centrifugation at 3200xg. 1:1 Chloroform extraction of the aqueous phase at 3200xg was followed by the addition of 1/40 volume 3 M sodium acetate solution and 2 volumes ethanol. Genomic DNA was precipitated at 16000xg and washed with 70% ethanol before resuspension of the air dried pellet in 10 mM Tris 1mM EDTA pH 7.5.   Following resuspension, equal amounts of genomic DNA from the five arsenate-treated animals were pooled together and equal amounts of genomic DNA from the five control animals were pooled together.  Five µg of pooled genomic DNA was digested with 400 U MspI (New England Biolabs, Ipswich, MA) in 100 µl at 37 °C for 18 - 24 hrs followed by QIAQuick purification (Qiagen, Valencia, CA). The end of digested DNA was filled in and an adenine was added to the 3’ end of the DNA fragments according to the Illumina standard end repair and add_A protocol (Illumina, San Diego, CA). Pre-annealed forked Illumina adaptors containing 5’-methyl-cytosine instead of cytosine was ligated to both ends of DNA fragments using standard Illumina adaptor ligation protocol (Illumina). Ligated fragments were then separated by 2% agarose gel (Sigma-Aldrich). Two size ranges, 150-175 bp and 175-225 bp (includes adaptor length), were selected and cut from the gel. DNA from gel slices were purified using Qiagen Gel extraction kit (Qiagen). The purified DNA was treated with EpiTect Bisulfite kit (Qiagen) with modification.  The bisulfite conversion time was extended to approximately 14 hrs by adding 3 cycles of denaturation at 95 °C for 5 min followed by incubation at 60 °C for 180 min. The bisulfite-converted DNA was purified using the EpiTect Bisulfite kit and protocol for DNA isolated from formaldehyde-fixed, paraffin-embedded tissue samples with modification.  The desulfonation buffer was added to the spin column and incubated for 20 min instead of 15 min at room temperature. The bisulfite-treated DNA was purified a second time with MinElute PCR purification kit (Qiagen) and eluted with 15 µl EB buffer. The bisulfite-treated DNA fragments were amplified using polymerase chain reaction (PCR) and the following reaction: 15 µl of eluted DNA, 5 pmol of Illumina PE PCR primers 1.0 and 2.0, 62.5 nM of each dNTP, and 2.5 U of Pfu Turbo Cx hotstart DNA polymerase (Stratagene Products, Agilent, La Jolla, CA) in a total 50 µl volume.  The amplification conditions were as follows: 5 min at 95 °C, 30 sec at 98 °C then 6X (10 sec at 98 °C, 30 sec at 65 °C, 30 sec at 72 °C) followed by 5 min at 72 °C. The PCR reaction was purified by MinElute PCR purification Kit (Qiagen) and final reduced representation bisulfite library was eluted in 15 µl EB. The concentration of final library was measured using the Agilent 2100 Bioanalyzer (Palo Alto, CA).  The library was sequenced on Illumina GA sequencing instrument according to standard Illumina cluster generation and sequencing protocols.