GSM3898233: 10h-IAA-14hR-rescue; Homo sapiens; Bisulfite-Seq
Harvest cells and extract DNA with phenol-chloroform. Fragment DNA with sonication (Covaris M200) or restrictive enzyme AluI (NEB, R0137). Size select DNA fragments ranging from 100 bp to 200 bp with AMPure beads (1.05X to 2.8X) (Beckman, A63881). Click it reaction to conjugate biotin to EdU labeled DNA: (Tris pH7.5 buffer 15 mM; Biotin azide (Sigma, 762024) 400 uM; CuSO4 (Sigma, 209198)/THPTA (Sigma, 762342) mix (1:5) (100 uM: 500 uM); Sodium ascorbate (freshly prepared, Sigma, A4034) 5 mM). Reaction at 34 °C for 20 min. DNA was purified with MinElute PCR purification kit (Qiagen, 28004). The click it reaction system is modified from (1) (2). End repair (NEB, E6050), dA-tailing (NEB, E6053) and adaptor ligation (NEB, E6056). Hairpin adapter and methylated sequencing adapter were pre-mix at the molar ratio of 3:1 before adapter ligation. After ligation, 1.4X AMPure beads were added to remove un-ligated adapters. Dissolve adapter ligated DNA in 1X TE buffer. Incubate DNA with M280 streptavidin beads (Thermo Fisher, 11205D) in 1X Binding & Washing buffer (5 mM Tris-Cl (pH 7.5), 1M NaCl, 0.5 mM EDTA (pH 8.0)) for 20 min. Wash with 1X Binding & Washing buffer containing 0.5% IGEPAL CA-630 (Sigma, I3021) for 6 times, and then wash with ddH2O once. Dissolve beads in 40 ul ddH2O. Perform bisulfite conversion (Qiagen, 59104) with DNA on beads. PCR amplification (KAPA KR0413) with bisulfite converted DNA. PCR product was running on a 2% agarose-gel and the larger band ranging from 300 bp to 400 bp was hairpin-ligated sample. Recover this band with gel-extraction kit (TIANGEN, DP208). Clean up with AMPure beads (0.9X). Dissolve DNA in 0.1X TE buffer. The HAMMER-seq library is ready for Next Generation Sequencing.
GEO
GSM3898233