GSM9350453: Gc13; Giraffa camelopardalis antiquorum; MeDIP-Seq
Genomic DNA was isolated from giraffe buccal cells using a protocol involving proteinase treatment, protein precipitation, and DNA precipitation in isopropanol. Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed. 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 ºC, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP (immunoprecipitation) 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 ºC. 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. 50 μl 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 ºC. 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 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol were added and mixed well, then precipitated at -20 ºC for 1 hour to overnight. The precipitate was centrifuged at 14,000 rpm for 20 min at 4 ºC and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol in a -20 ºC freezer for 15 min then centrifuged again at 14,000 rpm for 5 min at 4 ºC 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 a Qubit device (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.