GSM3357735: Dam- salmonella 6mACEseq; Salmonella enterica subsp. enterica serovar Typhimurium; OTHER
Bacteria were lysed overnight in SNET lysis buffer (400mM NaCl, 1% SDS, 20mM Tris pH 8, 5mM EDTA, 200µg/ml Proteinase K) at 56oC with shaking at 1,000rpm. Genomic DNA was purified from cell lysate using Phenol-chloroform-Isoamyalcohol (25:24:1), precipitated with 50% isopropanol. The DNA pellet was washed once with 100% ethanol and once with 70% ethanol before being reconstituted in IDTE (10mM Tris pH 8, 0.1mM EDTA). Genomic DNA was first sonicated to ~150bp and treated with RNAseA/T1 (Ambion AM2286) for 1hr at 37oC then purified using 1.8X volume Agencourt AMpure XP beads (Beckman A63880). 5µg of sonicated and RNA-free genomic DNA was then incubated with 4µg anti-6mA antibody (Synaptic Systems 202003) in 1X IP buffer (150mM NaCl, 10mM Tris pH 7.4, 0.1% IGEPAL) overnight at 4oC. This antibody-genomic DNA mixture was then crosslinked with 0.15J 254nm UV radiation six times before being mixed with 40µl BSA-blocked Protein-A-dynabeads (Life Technologies 10002). Beads bound with crosslinked genomic DNA were then washed with 500µl of the following buffers in this order: Wash buffer 1 (1M NaCl, 50mM HEPES-KOH pH 7.4, 1% Triton X-100, 0.1% Sodium Deoxycholate, 2mM EDTA), Wash buffer 2 (0.5M NaCl, 50mM HEPES-KOH pH 7.4, 1% IGEPAL, 0.1% Sodium Deoxycholate, 2mM EDTA), Wash buffer 3 (1% Sodium Deoxycholate, 25mM LiCl, 10mM Tris pH 8, 1% Triton X-100, 2mM EDTA), TE (10mM Tris pH 8, 1mM EDTA) and finally 10mM Tris pH 8. Genomic DNA was then subjected to progressive on-bead reactions as follows: End-repair with 6U T4 DNA polymerase (NEB M0203) at 12oC for 30min, end-phosphorylation with 30U T4 PNK (NEB M0201) and T4 DNA ligase buffer (NEB B0202) at 37oC for 30min, first adapter ligation with 1500U T4 DNA ligase (NEB M0202) and 202.5pmol first adapter at 25oC for 2hr, fill-in DNA synthesis with 20U Phi29 DNA polymerase (NEB M0269), a second round of end-phosphorylation using the same conditions, Lamda exonuclease digestion with 20U Lambda exonuclease (NEB M0262) at 37oC for 30min and finally RecJf exonuclease digestion with 90U RecJf (NEB M0264) at 37oC for 30min. The same bead-wash steps were carried out between each reaction. DNA was then eluted off beads with elution buffer (0.5% SDS, 0.2M NaCl, 25mM Tris pH 8, 2mM EDTA) and treated with 8,000U of proteinase K  (Thermo Scientific EO0491) overnight at 65oC with shaking at 1,000rpm. The eluted ssDNA was ethanol-precipitated before undergoing the following reactions as follows: Annealing of PG162 primer to the reverse adapter and primer extension with 20U Phi29 polymerase, second adapter ligation with 1000U T4 DNA ligase and 30.15pmol forward adapter, dsDNA purification with 1.7x volume Ampure XP beads, 18 cycles amplification with Phusion High-fidelity PCR mastermix (NEB M0530) and finally, primer-dimer removal with 0.8X volume AMpure XP beads before undergoing sequencing on the Illumina Miseq or Nextseq single-end platforms.
GEO
GSM3357735