GSM7734075: mesendoderm-Igf2_Igf2_dCTCFinICR-CUT&RUN-CTCF-BS10389A; Mus musculus; OTHER
Cells were dissociated using TryPE-Select, spun-down and media aspirate. CUT&RUN experiments were conducted using the CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assay kit (Cell Signaling Technology, #86652) following the manufacturer's protocol. For each reaction, 5 million cells were used. The CTCF antibody (Cell Signaling Technology, #2899S) was used at a dilution of 1/50 for overnight incubation at 4°C. DNA fragments were purified using the DNA Clean & Concentrator-5 Kit (Zymo, D4014) according to the manufacturer's procedures, and eluted in 15 µL. Up to 11.15 µL purified DNA was taken directly into end repair for library construction using the Illumina dsDNA protocol. 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.