SRX18313659 GSM6742929_r1 SRP408675 PRJNA903217 SRS15804321 GSM6742929 GSM6742929 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313660 GSM6742930_r1 SRP408675 PRJNA903217 SRS15804322 GSM6742930 GSM6742930 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313661 GSM6742931_r1 SRP408675 PRJNA903217 SRS15804323 GSM6742931 GSM6742931 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313662 GSM6742932_r1 SRP408675 PRJNA903217 SRS15804324 GSM6742932 GSM6742932 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313663 GSM6742933_r1 SRP408675 PRJNA903217 SRS15804325 GSM6742933 GSM6742933 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313664 GSM6742934_r1 SRP408675 PRJNA903217 SRS15804326 GSM6742934 GSM6742934 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313665 GSM6742935_r1 SRP408675 PRJNA903217 SRS15804327 GSM6742935 GSM6742935 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313666 GSM6742936_r1 SRP408675 PRJNA903217 SRS15804328 GSM6742936 GSM6742936 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313667 GSM6742937_r1 SRP408675 PRJNA903217 SRS15804329 GSM6742937 GSM6742937 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313668 GSM6742938_r1 SRP408675 PRJNA903217 SRS15804330 GSM6742938 GSM6742938 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313669 GSM6742939_r1 SRP408675 PRJNA903217 SRS15804331 GSM6742939 GSM6742939 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313670 GSM6742940_r1 SRP408675 PRJNA903217 SRS15804332 GSM6742940 GSM6742940 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313671 GSM6742941_r1 SRP408675 PRJNA903217 SRS15804333 GSM6742941 GSM6742941 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313672 GSM6742942_r1 SRP408675 PRJNA903217 SRS15804334 GSM6742942 GSM6742942 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313673 GSM6742943_r1 SRP408675 PRJNA903217 SRS15804335 GSM6742943 GSM6742943 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313674 GSM6742944_r1 SRP408675 PRJNA903217 SRS15804336 GSM6742944 GSM6742944 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313675 GSM6742945_r1 SRP408675 PRJNA903217 SRS15804337 GSM6742945 GSM6742945 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313676 GSM6742946_r1 SRP408675 PRJNA903217 SRS15804338 GSM6742946 GSM6742946 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313677 GSM6742947_r1 SRP408675 PRJNA903217 SRS15804339 GSM6742947 GSM6742947 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313678 GSM6742948_r1 SRP408675 PRJNA903217 SRS15804340 GSM6742948 GSM6742948 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313679 GSM6742949_r1 SRP408675 PRJNA903217 SRS15804341 GSM6742949 GSM6742949 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313680 GSM6742950_r1 SRP408675 PRJNA903217 SRS15804342 GSM6742950 GSM6742950 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313681 GSM6742951_r1 SRP408675 PRJNA903217 SRS15804343 GSM6742951 GSM6742951 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313682 GSM6742952_r1 SRP408675 PRJNA903217 SRS15804344 GSM6742952 GSM6742952 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313683 GSM6742953_r1 SRP408675 PRJNA903217 SRS15804345 GSM6742953 GSM6742953 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313684 GSM6742954_r1 SRP408675 PRJNA903217 SRS15804346 GSM6742954 GSM6742954 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313685 GSM6742955_r1 SRP408675 PRJNA903217 SRS15804347 GSM6742955 GSM6742955 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313686 GSM6742956_r1 SRP408675 PRJNA903217 SRS15804348 GSM6742956 GSM6742956 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313687 GSM6742957_r1 SRP408675 PRJNA903217 SRS15804349 GSM6742957 GSM6742957 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313688 GSM6742958_r1 SRP408675 PRJNA903217 SRS15804350 GSM6742958 GSM6742958 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313689 GSM6742959_r1 SRP408675 PRJNA903217 SRS15804351 GSM6742959 GSM6742959 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313690 GSM6742960_r1 SRP408675 PRJNA903217 SRS15804352 GSM6742960 GSM6742960 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313691 GSM6742961_r1 SRP408675 PRJNA903217 SRS15804353 GSM6742961 GSM6742961 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313692 GSM6742962_r1 SRP408675 PRJNA903217 SRS15804354 GSM6742962 GSM6742962 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313693 GSM6742963_r1 SRP408675 PRJNA903217 SRS15804355 GSM6742963 GSM6742963 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313694 GSM6742964_r1 SRP408675 PRJNA903217 SRS15804356 GSM6742964 GSM6742964 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313695 GSM6742965_r1 SRP408675 PRJNA903217 SRS15804357 GSM6742965 GSM6742965 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313696 GSM6742966_r1 SRP408675 PRJNA903217 SRS15804358 GSM6742966 GSM6742966 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313697 GSM6742967_r1 SRP408675 PRJNA903217 SRS15804359 GSM6742967 GSM6742967 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313698 GSM6742968_r1 SRP408675 PRJNA903217 SRS15804360 GSM6742968 GSM6742968 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313699 GSM6742969_r1 SRP408675 PRJNA903217 SRS15804361 GSM6742969 GSM6742969 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313700 GSM6742970_r1 SRP408675 PRJNA903217 SRS15804362 GSM6742970 GSM6742970 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313701 GSM6742971_r1 SRP408675 PRJNA903217 SRS15804363 GSM6742971 GSM6742971 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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 SRX18313702 GSM6742972_r1 SRP408675 PRJNA903217 SRS15804364 GSM6742972 GSM6742972 MeDIP-Seq GENOMIC 5-methylcytidine antibody Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual 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 (10mM Tris HCl, pH7.5; 1mM 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 1XPBS with 0.1% BSA and 2mM 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 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl 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 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/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 14,000rpm 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 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm 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 20μl 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