title: GSM8755746: IP_CTCF_CTCF-Nm_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: 20 million cells were crosslinked in 37o prewarmed culture medium with 1% formaldehyde for 10 min at RT.  Cells were then treated with cell lysis buffer (5mM PIPES pH 8, 85mM KCl, 0.5% NP-40) for 10 min on ice, followed by treatment with nuclei lysis buffer (50mM TrisCl pH 8.1, 10mM EDTA, 1% SDS) for 10 min at RT. Chromatin was subjected to sonication with Diagenode Bioruptor at 4o to achieve an average size of 200-300 bp (30 sec on, 30 sec off, 20 cycles with high energy input). Chromatin were then precleared with Dynabeads Protein A at 4o for 2 hours. 1/30 lysates were kept as input and the rest incubated with 2.5ug SMC3 antibody  or CTCF antibody overnight at 4o. IP samples were then captured by Dynabeads Protein A at 4o for at least 2 hours, followed by bead washing and elution. IP and Input DNA were then de-crosslinked at 65o overnight and purified via Qiagen PCR purification columns. Purified DNA was subjected to ChIP-Seq library preparation with the NEBNext Ultra II DNA Library Prep Kit for Illumina from New England Biolabs and sequenced via paired end 150-bp sequencing on a Illumina NovoSeq 6000 machine.