title: GSM5639871: nANW7; Homo sapiens; MeDIP-Seq
library_construction_protocol: Buccal samples were frozen and stored (-20 °C) for the subsequent epigenetic analysis. Blood was collected into BD Vacutainer CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) by ANW Spokane, WA and shipped overnight on ice for immediate processing. Monocytes were isolated using antibody-linked magnetic beads (Dynabeads CD14, Life Technologies # 11149D). Human buccal samples were kept frozen and thawed for analysis. Blood samples were kept refrigerated before monocyte isolation. For monocyte isolation the blood was diluted 1:2 in Isolation Buffer (1X PBS, 0.1% BSA, 2mM EDTA) then centrifuged at 600 x g for 10 min at room temperature in a swinging bucket rotor. The plasma upper layer was discarded, and the blood resuspended to the original volume in Isolation Buffer. Magnetic beads (Dynabeads CD14, Life Technologies # 11149D) were added to the blood samples (25 µL of pre-washed beads for 1 ml of blood sample). The mixture (beads-cells) was incubated for 20 min between 2 °C and 8 °C with gentle tilting and rotation. The mixture was then placed on a magnet for 2 minutes and the supernatant carefully removed. One ml of Isolation Buffer was added to the beads-cells mixture then homogenized and incubated 2 minutes on the magnet. This step was repeated at least 3 times. The bead-bound cells were resuspended in 100 µl 1X PBS pH 7.4 and genomic DNA isolation was performed.  Genomic DNA from buccal swabs or monocytes was prepared as follows: The buccal swabs were incubated with 750 µl of cell lysis solution (100 mM NaCl, 10 mM Tris, 25 mM EDTA, 0.5% SDS) and 3.5 µl of Proteinase K (20 mg/ml) for 3 hours in a heating block at 55 °C. The lysis solution was transferred to a new tube and spun for 2 min at full speed. For the monocytes, 820 μl of DNA extraction buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.5% SDS) and 80 μl Proteinase K (20 mg/ml) were added to the bead-bound monocytes and the sample incubated at 55 °C for 2 hours under constant rotation. The samples were placed on a magnet where the magnetic beads were discarded and the supernatants containing the DNA kept. Then for both the buccal swabs and the monocytes, 300 μl of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A, Madison, WI) was added, the sample was mixed and incubated on ice for 15 min, then spun at 4 °C at 13,500 x g for 20 min. The supernatant was transferred to a fresh tube, then precipitated over night at -20 °C with 1 mL of cold 100% isopropanol and 2 μl glycoblue. The sample was then centrifuged at 4 °C, 13,500 x g for 20 min. The supernatant was discarded and the pellet washed with 70% cold ethanol then returned to freezer for 20-30 min. The sample was then centrifuged at 4 °C, 13,500 x g for 10 min. The supernatant was discarded, and the pellet air-dried for 5 min then resuspended in 100 μl nuclease free water. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual genomic DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1X Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to 400 μl, heat-denatured for 10 min at 95 °C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 °C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml 1X PBS with 0.1% BSA and 2 mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1X IP buffer (50 mM sodium phosphate pH 7.0, 700 mM NaCl, 0.25% Triton X-100) as the initial volume of beads. Beads (50 μl) were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 °C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1X IP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20 mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 °C, then 250 μl of buffered Phenol-Chloroform-Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000 x g for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 13,500 x g for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated in -20 °C freezer for 1 hour to overnight. The precipitate was centrifuged at 13,500 x g for 20 min at 4 °C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in -20 °C freezer for 15 min then centrifuged again at 13,500 x g for 5 min at 4 °C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212). The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA. After this step the manufacturer's protocol was followed. Each sample received a separate index primer.