GSM5963866: ESC, TET-TKO, TET3, Ctr 0h, B, 5hmC; Mus musculus; OTHER
Total nucleic acids were purified using the Bio-On-Magnetic-Beads (BOMB) approach. Briefly, a guanidine isothiocyanate lysis buffer was used to homogenize cells, and then was combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in volumetric ratio of 2:3:4 (beads:lysate:isopropanol). Beads were captured with a neodymium magnet and washed once with isopropanol, twice with 70% ethanol and resuspended in milliQ water. For ACE-seq, 10 ng of purified DNA was enzymatically fragmented to ~300 bp with dsDNA Fragmentase (#M0348 NEB), according to the manufacturer instructions, with the reaction scaled down 50%, and incubation time of 37 °C for 32 min. Fragmented DNA was concentrated using PEG-diluted SPRI beads. In a total volume of 5 μl, 5hmC was glucosylated with 5 U T4- βGT in NEB buffer 4 supplemented with 2 mM UDP-Glucose at 37 °C for 1 h. Glucosylated DNA was denatured in the presence of 20% DMSO at 95 °C for 5 min then swiftly transferred to a pre-cooled PCR tube rack on dry ice for snap-cooling. The single stranded glucosylated DNA was then deaminated in 50 mM Bis-Tris (pH 6.0), 0.1% Triton X-100 (APOBEC Reaction Buffer, EM-seq component E7134AA, NEB), 20 µg BSA (NEB) using 0.2 µg of APOBEC3A (EM-seq component E7133AA, NEB), in a total volume of 10 μl. DNA was deaminated by APOPBEC3A at 4 °C for 10 minutes, then ramping from 4 to 50 °C over 2 h followed by 50 °C for 10 min as previously reported (46). First strand synthesis, second strand synthesis for adapter tagging, and PCR, was completed as described above for PBAT. Library integrity was determined by agarose gel electrophoresis, libraries were pooled and size selected by gel extraction (MinElute® Gel Extraction Kit, Qiagen) and sequenced over 3 Illumina iSeq100 runs using 150bp chemistry. APOBEC-coupled epigenetic sequencing (ACE-seq)