GSM7746907: L528-IgG; Homo sapiens; Breviolum minutum; MeDIP-Seq
B. minutum genomic DNA was isolated as follows. Briefly, cells were centrifuged at 1,000 g for 5 minutes, then resuspended in 500 µL 1× Cell Lysis Buffer (prepared by mixing equal volumes of 2× Cell Lysis Buffer – 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl, pH 8.0 – and H2O) and vortexed. The lysed cells were mixed with an equal 500 µL volume Phenol:Chloroform:Isoamyl alcohol (25:24:1), and mixed well by inverting a few times. The phases were centrifugation at 13,000 g for 5 minutes, then the top phase was transferred to a new tube and treated with 4 µL Ribonuclease A (20 mg/mL) by incubating for 30 minutes at 37◦C. DNA was purified by adding an equal volume Phenol:Chloroform:Isoamyl alcohol (25:24:1), mixing well and centrifuging at 13,000 g for 5 minutes, then transferring the top layer to a new tube, to which Phenol:Chloroform:Isoamyl alcohol (25:24:1) was added again, and the centrifugation and top phase isolation was repeated. Then 2.5× volumes of 100% EtOH were added and the mixture was incubated on ice for 30 minutes or at -20◦C overnight. The solution was then centrifuged at 13,000 g at room temperature for 20 minutes, the pellet was washed with 70% EtOH, dried on air and resuspended in 50 µL H2O. To prepare inputs for MeDIP-seq experiments, gDNA was first sonicated using a Qsonica S-4000 with a 1/16” tip for 3 minutes, with 10 second pulses at intensity 3.5, and 20 seconds rest between pulses. For each reaction, 100 µL of Protein A Dynabeads (ThermoFisher Cat # 10002D) were washed 3 times with a 5 mg/mL BSA solution. Beads were then resuspended in 1 mL BSA solution and 5 µL of α-5-hmU antibody (Abcam Cat # ab19735) were added. Coupling of antibodies to beads was carried out overnight on a rotator at 4◦C. Beads were again washed 3 times with BSA solution and resuspended in 100 µL of BSA solution. Sheared genomic DNA (∼1 µg 1:1 mix of B. minutum and Homo sapiens) was end repaired and adapters were ligated to it following the procedure of the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S), purified using AMPure XP beads and eluted in 50 µL of H2O, and then denatured at 98◦C for 10 minutes. DNA was then immediately placed on ice, resuspended in 850 µL RIPA buffer (1× PBS, 1% IGEPAL, 0.5% Sodium Deoxycholate, 0.1% SDS, Roche Protease Inhibitor Cocktail) and added to the beads, then incubated overnight on a rotator at 4◦C. Beads were washed 5 times with LiCl buffer (10 mM Tris-HCl pH 7.5, 500 mM LiCl, 1% NP-40/IGEPAL, 0.5% Sodium Deoxycholate) by incubating for 10 minutes at 4◦C on a rotator, then rinsed once with 1× TE buffer. Beads were then resuspended in 200 µL IP Elution Buffer (1% SDS, 0.1 M NaHCO3) and incubated at 65◦C in a Thermomixer (Eppendorf) with interval mixing to dissociate antibodies. Beads were separated from the DNA solution by centrifugation, and DNA was purified using the MinElute kit. Library generation was completed by carrying out PCR following the rest of the steps of the NEBNext Ultra II DNA Library Prep Kit protocol, using 15 cycles of amplification. Final libraries were purified using AMPure XP beads.