GSM7851673: mESC E14, nuclei, m6dA methylated, ex situ SAMOSA-Tag; Mus musculus; OTHER
Tagmented nuclei were pre-treated with 10uL RNaseA at 37°C for 15 minutes, then purified with 2.65uL Proteinase K (20mg/mL), and 2.65uL 10% SDS at 65°C for 3 hours. DNA was extracted using an equal volume of phenol:chloroform:isoamyl Alcohol (25:24:1, v/v), followed by 100% ethanol percipitation overnight at -80ºC. Extracted DNA was tagmented using SMRT-Tag conditions (in 1X Tagmentation Mix (10mM TAPS-NaOH pH 8.5, 5mM MgCl2, and 10% DMF) , 55ºC for 30 min) with 0.046 pmol H-Tn5-R27S,E54K,L372P (QB3 MarcoLab) , then terminated  by adding 0.2% SDS (final concentration 0.04%). Tagmented fragments were gap-repaired using Phusion DNA polymerase and Taq Ligase at 37ºC for 1hr, digested with Exonuclease III at 37ºC for 1hr, then cleaned up using Ampure PB. Library quality was assessed with Qubit 1x High Sensitivity DNA Assay and Agilent Bioanalyzer High Sensitivity DNA kit. OTHER: Ex situ SAMOSA-Tag Library Preparation