title: GSM8126193: 6mA-ChIP-exo hypoxia 2; Homo sapiens; OTHER
library_construction_protocol: DNA was extracted from cell pellets using the phenol chloroform, followed by DNA fragmentation in the range of 100-200 bp according to the manufacturer's protocol for the Bioruptor Pico with 0.65 ml microtubes. 30~100 µg of fragmented DNA was hybridized with 3 µg of the 6mA antibody (sysy-003, Synapse) overnight. The DNA:antibody mixture was crosslinked using UVC irradiation in 1× IP buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Igepal-630) and pulled down by protein A dynabeads (Invitrogen). Subsequent washes were performed with FA lysis buffer (50 mM HEPES-KOH pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), NaCl buffer (50 mM HEPES-KOH pH 7.4, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl buffer (100 mM Tris-HCl pH 7.9, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate), TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA), and Tris buffer (10 mM Tris-HCl pH 7.4) in following enzymatic steps: DNA repair with PreCR Repair mix (M0309S, NEB), A-taling with Klenow Fragment (3'-5', exo-) (M0212S, NEB), ligase the adapter with T4 ligase (M0202S, NEB) and T4 Polynucleotide Kinase (M0201S, NEB), fill-in with phi29 DNA polymerase (M0269S, NEB), and digest with lambda exonuclease (M0262S, NEB). After a series of enzymatic reactions, DNA was eluted using an elution buffer (100 mM NaHCO3, 1% SDS) combined with RNase A. The sample was transferred to a new tube and digested with proteinase K at 56°C overnight. DNA was purified using 3× SPRI beads (Beckman) clean-up, followed by a second adapter ligation using a splint adapter. The adapter ligated sample was purified by 3× SPRI beads clean-up. Amplification was performed using 1× Phusion High-Fidelity PCR Master Mix (NEB) and Illumina library primers (P-1.3 and P-2.1) for 12-14 cycles. The product was then purified using 1× SPRI beads, resulting in a sequence library with an approximate concentration of 1~2 nM.