WT(SB210) 6mA IP of Tetrahymena
Isolated genomic DNA was diluted to 100-200 ng/uL using TE buffer, and sonicated in 130 uL scale to 200-400 bp using a Covaris Focused-ultrasonicator. End repair, 3-adenylation, and adapter ligation were performed. The ligated and purified DNA was denatured at 95 C, and chilled on ice. A portion of 10 uL DNA was saved as input. The rest of the DNA was combined with 3 ug of anti-6mA antibody (Synaptic Systems) in 500 uL of 1 IP buffer, and incubated at 4 C for 6 hr. At the same time, 40 uL of Protein A magnetic beads was washed twice in 0.5 mL of 1 IP buffer, and pre-blocked in 0.5 mL of 1 IP buffer containing 20 ug/uL of Bovine Serum Album at 4 C for 6 hr. The Protein A beads were washed twice with 0.5 mL of 1 IP buffer, added to the DNA-6mA antibody mixture, and incubated at 4 C with gentle rotation overnight. The beads were then washed four times with 0.5 mL of 1 IP buffer. Methylated DNA was eluted twice by 100 uL of elution buffer containing 6mA monophosphate at 4 C for 1 hr. The two elusion solutions were combined, to which 20 uL of NaOAc (3 M, pH 5.3), 500 uL of EtOH, and 0.5 uL of glycogen (20 ng/uL) were added. The solution was frozen at 80 C overnight, and centrifuged at 14,000g for 20 min at 4 C. The precipitated DNA was dissolved in 7 uL of ddH2O, PCR amplified for 15 to 18 cycles, purified by Ampure beads, and suspended in 16 uL of re-suspension buffer to yield the sequencing library.