title: GSM9375593: Micro-C-TALE_rsCBE_rep1; Mus musculus; OTHER
library_construction_protocol: Approximately 1.6–4 × 10⁶ CD19⁺ pre-B cells were enriched from WT-Rag1⁻/⁻.hIgM and ΔrsCBE-Rag1⁻/⁻.hIgM mice using anti-CD19 magnetic beads (Miltenyi Biotec) and MACS column separation (Miltenyi Biotec). Fixed and permeabilized cells were treated with micrococcal nuclease (MNase) (ThermoFisher, #88216) to obtain predominantly mononucleosomal DNA. The DNA was then ligated using T4 DNA ligase (NEB, #M0202L), extracted by phenol–chloroform and ethanol precipitation, and ligation products were enriched by size selection using Ampure XP beads (Beckman Coulter, #A63881). End repair and adaptor ligation were performed using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645S), followed by PCR amplification with indexed primers. Four Micro-C libraries were generated and pooled in equimolar proportions. Capture probes were prepared from fifteen BACs spanning the target genomic region. BAC DNA was mixed equimolarly, sheared to ~200-bp fragments by sonication, end-repaired, and ligated to specialized adaptors using the NEBNext Ultra II kit. The BAC library was then PCR-amplified using biotinylated primers. Hybridization-based capture was performed by incubating the pooled Micro-C libraries with the biotinylated BAC-derived probes in multiple replicates. Hybridized products were pulled down using streptavidin-coated magnetic beads (Dynabeads MyOne sterptavidin C1, Invitrogen, #65001) and amplified by PCR with Illumina-compatible primers. The capture cycle was repeated once, and the final libraries were sequenced on a DNBSEQ-G400 machine using paired-end 150-bp reads.