SRX12694426 GSM5639829 SRP342182 SRS10645293 GSM5639829 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639829 GSM5639829 GEO Accession GSM5639829 SRX12694427 GSM5639830 SRP342182 SRS10645294 GSM5639830 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639830 GSM5639830 GEO Accession GSM5639830 SRX12694428 GSM5639831 SRP342182 SRS10645295 GSM5639831 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639831 GSM5639831 GEO Accession GSM5639831 SRX12694429 GSM5639832 SRP342182 SRS10645296 GSM5639832 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639832 GSM5639832 GEO Accession GSM5639832 SRX12694430 GSM5639833 SRP342182 SRS10645297 GSM5639833 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639833 GSM5639833 GEO Accession GSM5639833 SRX12694431 GSM5639834 SRP342182 SRS10645298 GSM5639834 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639834 GSM5639834 GEO Accession GSM5639834 SRX12694432 GSM5639835 SRP342182 SRS10645299 GSM5639835 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639835 GSM5639835 GEO Accession GSM5639835 SRX12694433 GSM5639836 SRP342182 SRS10645300 GSM5639836 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639836 GSM5639836 GEO Accession GSM5639836 SRX12694434 GSM5639837 SRP342182 SRS10645301 GSM5639837 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639837 GSM5639837 GEO Accession GSM5639837 SRX12694435 GSM5639838 SRP342182 SRS10645302 GSM5639838 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639838 GSM5639838 GEO Accession GSM5639838 SRX12694436 GSM5639839 SRP342182 SRS10645303 GSM5639839 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639839 GSM5639839 GEO Accession GSM5639839 SRX12694437 GSM5639840 SRP342182 SRS10645304 GSM5639840 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639840 GSM5639840 GEO Accession GSM5639840 SRX12694438 GSM5639841 SRP342182 SRS10645305 GSM5639841 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639841 GSM5639841 GEO Accession GSM5639841 SRX12694439 GSM5639842 SRP342182 SRS10645306 GSM5639842 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639842 GSM5639842 GEO Accession GSM5639842 SRX12694440 GSM5639843 SRP342182 SRS10645307 GSM5639843 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639843 GSM5639843 GEO Accession GSM5639843 SRX12694441 GSM5639844 SRP342182 SRS10645308 GSM5639844 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639844 GSM5639844 GEO Accession GSM5639844 SRX12694442 GSM5639845 SRP342182 SRS10645309 GSM5639845 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639845 GSM5639845 GEO Accession GSM5639845 SRX12694443 GSM5639846 SRP342182 SRS10645310 GSM5639846 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639846 GSM5639846 GEO Accession GSM5639846 SRX12694444 GSM5639847 SRP342182 SRS10645311 GSM5639847 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639847 GSM5639847 GEO Accession GSM5639847 SRX12694445 GSM5639848 SRP342182 SRS10645312 GSM5639848 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639848 GSM5639848 GEO Accession GSM5639848 SRX12694446 GSM5639849 SRP342182 SRS10645313 GSM5639849 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639849 GSM5639849 GEO Accession GSM5639849 SRX12694447 GSM5639850 SRP342182 SRS10645314 GSM5639850 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639850 GSM5639850 GEO Accession GSM5639850 SRX12694448 GSM5639851 SRP342182 SRS10645315 GSM5639851 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639851 GSM5639851 GEO Accession GSM5639851 SRX12694449 GSM5639852 SRP342182 SRS10645316 GSM5639852 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639852 GSM5639852 GEO Accession GSM5639852 SRX12694450 GSM5639853 SRP342182 SRS10645317 GSM5639853 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639853 GSM5639853 GEO Accession GSM5639853 SRX12694451 GSM5639854 SRP342182 SRS10645318 GSM5639854 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639854 GSM5639854 GEO Accession GSM5639854 SRX12694452 GSM5639855 SRP342182 SRS10645319 GSM5639855 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639855 GSM5639855 GEO Accession GSM5639855 SRX12694453 GSM5639856 SRP342182 SRS10645320 GSM5639856 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639856 GSM5639856 GEO Accession GSM5639856 SRX12694454 GSM5639857 SRP342182 SRS10645321 GSM5639857 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639857 GSM5639857 GEO Accession GSM5639857 SRX12694455 GSM5639858 SRP342182 SRS10645322 GSM5639858 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639858 GSM5639858 GEO Accession GSM5639858 SRX12694456 GSM5639859 SRP342182 SRS10645323 GSM5639859 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639859 GSM5639859 GEO Accession GSM5639859 SRX12694457 GSM5639860 SRP342182 SRS10645324 GSM5639860 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639860 GSM5639860 GEO Accession GSM5639860 SRX12694458 GSM5639861 SRP342182 SRS10645325 GSM5639861 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639861 GSM5639861 GEO Accession GSM5639861 SRX12694459 GSM5639862 SRP342182 SRS10645326 GSM5639862 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639862 GSM5639862 GEO Accession GSM5639862 SRX12694460 GSM5639863 SRP342182 SRS10645327 GSM5639863 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639863 GSM5639863 GEO Accession GSM5639863 SRX12694461 GSM5639864 SRP342182 SRS10645328 GSM5639864 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639864 GSM5639864 GEO Accession GSM5639864 SRX12694462 GSM5639865 SRP342182 SRS10645329 GSM5639865 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639865 GSM5639865 GEO Accession GSM5639865 SRX12694463 GSM5639866 SRP342182 SRS10645330 GSM5639866 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639866 GSM5639866 GEO Accession GSM5639866 SRX12694464 GSM5639867 SRP342182 SRS10645331 GSM5639867 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639867 GSM5639867 GEO Accession GSM5639867 SRX12694465 GSM5639868 SRP342182 SRS10645332 GSM5639868 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639868 GSM5639868 GEO Accession GSM5639868 SRX12694466 GSM5639869 SRP342182 SRS10645333 GSM5639869 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639869 GSM5639869 GEO Accession GSM5639869 SRX12694467 GSM5639870 SRP342182 SRS10645334 GSM5639870 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639870 GSM5639870 GEO Accession GSM5639870 SRX12694468 GSM5639871 SRP342182 SRS10645335 GSM5639871 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639871 GSM5639871 GEO Accession GSM5639871 SRX12694469 GSM5639872 SRP342182 SRS10645336 GSM5639872 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639872 GSM5639872 GEO Accession GSM5639872 SRX12694470 GSM5639873 SRP342182 SRS10645337 GSM5639873 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639873 GSM5639873 GEO Accession GSM5639873 SRX12694471 GSM5639874 SRP342182 SRS10645338 GSM5639874 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639874 GSM5639874 GEO Accession GSM5639874 SRX12694472 GSM5639875 SRP342182 SRS10645339 GSM5639875 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639875 GSM5639875 GEO Accession GSM5639875 SRX12694473 GSM5639876 SRP342182 SRS10645340 GSM5639876 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639876 GSM5639876 GEO Accession GSM5639876 SRX12694474 GSM5639877 SRP342182 SRS10645341 GSM5639877 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639877 GSM5639877 GEO Accession GSM5639877 SRX12694475 GSM5639878 SRP342182 SRS10645342 GSM5639878 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639878 GSM5639878 GEO Accession GSM5639878 SRX12694476 GSM5639879 SRP342182 SRS10645343 GSM5639879 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639879 GSM5639879 GEO Accession GSM5639879 SRX12694477 GSM5639880 SRP342182 SRS10645344 GSM5639880 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639880 GSM5639880 GEO Accession GSM5639880 SRX12694478 GSM5639881 SRP342182 SRS10645345 GSM5639881 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639881 GSM5639881 GEO Accession GSM5639881 SRX12694479 GSM5639882 SRP342182 SRS10645346 GSM5639882 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639882 GSM5639882 GEO Accession GSM5639882 SRX12694480 GSM5639883 SRP342182 SRS10645347 GSM5639883 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639883 GSM5639883 GEO Accession GSM5639883 SRX12694481 GSM5639884 SRP342182 SRS10645348 GSM5639884 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639884 GSM5639884 GEO Accession GSM5639884 SRX12694482 GSM5639885 SRP342182 SRS10645349 GSM5639885 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639885 GSM5639885 GEO Accession GSM5639885 SRX12694483 GSM5639886 SRP342182 SRS10645350 GSM5639886 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639886 GSM5639886 GEO Accession GSM5639886 SRX12694484 GSM5639887 SRP342182 SRS10645351 GSM5639887 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639887 GSM5639887 GEO Accession GSM5639887 SRX12694485 GSM5639888 SRP342182 SRS10645352 GSM5639888 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639888 GSM5639888 GEO Accession GSM5639888 SRX12694486 GSM5639889 SRP342182 SRS10645353 GSM5639889 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639889 GSM5639889 GEO Accession GSM5639889 SRX12694487 GSM5639890 SRP342182 SRS10645354 GSM5639890 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639890 GSM5639890 GEO Accession GSM5639890 SRX12694488 GSM5639891 SRP342182 SRS10645355 GSM5639891 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639891 GSM5639891 GEO Accession GSM5639891 SRX12694489 GSM5639892 SRP342182 SRS10645356 GSM5639892 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639892 GSM5639892 GEO Accession GSM5639892 SRX12694490 GSM5639893 SRP342182 SRS10645357 GSM5639893 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639893 GSM5639893 GEO Accession GSM5639893 SRX12694491 GSM5639894 SRP342182 SRS10645358 GSM5639894 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639894 GSM5639894 GEO Accession GSM5639894 SRX12694492 GSM5639895 SRP342182 SRS10645359 GSM5639895 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639895 GSM5639895 GEO Accession GSM5639895 SRX12694493 GSM5639896 SRP342182 SRS10645360 GSM5639896 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639896 GSM5639896 GEO Accession GSM5639896 SRX12694494 GSM5639897 SRP342182 SRS10645361 GSM5639897 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639897 GSM5639897 GEO Accession GSM5639897 SRX12694495 GSM5639898 SRP342182 SRS10645362 GSM5639898 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639898 GSM5639898 GEO Accession GSM5639898 SRX12694496 GSM5639899 SRP342182 SRS10645363 GSM5639899 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639899 GSM5639899 GEO Accession GSM5639899 SRX12694497 GSM5639900 SRP342182 SRS10645364 GSM5639900 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639900 GSM5639900 GEO Accession GSM5639900 SRX12694498 GSM5639901 SRP342182 SRS10645365 GSM5639901 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639901 GSM5639901 GEO Accession GSM5639901 SRX12694499 GSM5639902 SRP342182 SRS10645366 GSM5639902 MeDIP-Seq GENOMIC 5-methylcytidine antibody Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed. Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer. Illumina HiSeq 2500 gds 305639902 GSM5639902 GEO Accession GSM5639902