GSM8987461: LP-161kb_Sox2P_mChDel_CM2266_rep2; Mus musculus; OTHER
Region-capture Micro-C (RCMC) was performed as previously described (Goel et al. 2023 PMID: 37157000). Briefly, nuclei were extracted from crosslinked cells and the chromatin digested with micrococcal nuclease into nucleosome-sized fragments. These fragments were labeled with biotinylated nucleotides and ligated by T4 DNA ligase to other spatially proximal fragments overnight. Crosslinks were reversed and dinucleosome-sized fragments were selected by electrophoresis on a 1% agarose gel and purified by pulldown on T1 streptavidin beads (Invitrogen). Purified dinucleosome-sized fragments were prepared for Illumina sequencing using an NEBNext Ultra II library prep kit (NEB #E7645S) following manufacturer's instructions. Prepared libraries on beads were amplified by PCR using Q5 DNA polymerase to generate sufficient input for target enrichment. Prior to sequencing, the target region was enriched via binding to biotinylated DNA probes and streptavidin bead pulldown (Twist). The enriched library was PCR amplified using Equinox library amplification mix; the minimum number of cycles to generate sufficient input for sequencing was performed. Libraries were sequenced on an Illumina NovaSeq X.