GSM5575015: mNPC_WAPLAID_ChIP_CTCF_24h; Mus musculus; ChIP-Seq
DamID: Genomic DNA was isolated using the Bioline Isolate II Genomic DNA Kit. The DNA concentration was estimated with a Nanodrop; ChIP-seq: ChIP experiments were performed in the presence of 10% human HEK293T cells as internal reference. Cells were crosslinked (1% FA, 10 minutes) and quenced with glycine (2.0M). Chromatin was fragmented to ~300 bp fragments with a Bioruptor Plus sonication device (Diagenode). CTCF antibody (07-729, Merck Millipore; 5 μL per ChIP) was coupled with Protein G beads (Thermo Fisher Scientific) and incubated with fragmented chromatin overnight at 4 °C. Released DNA fragments were purified with the MinElute PCR Purification kit (Qiagen). DamID: The full protocol is described in Leemans, 2019. 500 ng of genomic DNA was digested with DpnI at Dam-methylated GATC sequences, and ligated to adapters. Subsequent DpnII digestion destroyed DNA fragments containing unmethylated GATC sequences. DNA fragments were then amplified with 15-22 PCR cycles. Amplified fragments were further processed with end-repair, 3A-overhang tailing and Y-shaped adapter ligation, before a final round of 10-12 PCR cycles with illumina index adapters; ChIP-seq: The purified DNA fragments were prepared with the KAPA HTP Library Preparation kit (Roche).