GSM7734209: mesendoderm-DNase-BS03687A; Mus musculus; OTHER
Cells were dissociated using TryPE-Select, spun-down and media aspirate. DNase digestion was performed as described previously (John et al. 2013; Georgolopoulos et al. 2021) and adapted to 200 µL thermocycler tubes. Briefly, nuclei were extracted from cells and incubated with limiting concentrations of the DNA endonuclease DNase I (Sigma D4527-20KU) supplemented with Ca2+ and Mg2+ for 3 min at 37 °C. Digestion was stopped by the addition of EDTA, and the samples were treated with proteinase K and RNase A. Short double-hit fragments were isolated from DNaseI digestion using magnetic bead polyethylene glycol (PEG) fractionation. DNA fragments were end-repaired with T4 DNA polymerase (NEB M0203L), Klenow DNA polymerase (NEB M0210L), and T4 polynucleotide kinase (NEB M0201L), and A-tailed using Klenow (3′-5′ exo-; NEB M0212L). Illumina sequencing adapters were then ligated to DNA ends using Quick Ligase (NEB M2200L). The post-ligation product was purified using 18% Sera-Mag Magnetic Beads (Cytiva 65152105050250) in polyethylene glycol. DNA libraries were amplified with KAPA 2× Hi-Fi Hotstart Readymix (Roche 07958935001) and purified with 18% Sera-Mag Magnetic Beads in polyethylene glycol.