title: GSM8685732: WT BMDM, untreated, replicate2 (MNase-seq); Mus musculus; MNase-Seq
library_name: GSM8685732
library_construction_protocol: Cells were resuspended in a lysis buffer containing 15 mM NaCl, 15 mM Tris-HCl (pH 7.6), 60 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.3 M sucrose, 0.5 mM PMSF, 1 mM DTT, 0.2 mM spermine, and 1 mM spermidine. Lysis was achieved by adding 0.4% NP-40, and nuclei were collected and washed to remove detergent traces. MNase digestion was performed in a buffer containing 20 mM Tris-HCl (pH 7.6) and 5 mM CaCl₂ using 1.3 U MNase (Roche Cat# 10107921001) per sample. The reaction was incubated at 37°C for 100 minutes with gentle shaking, and digestion was stopped with 50 mM EDTA. Mononucleosomal DNA was isolated using gel electrophoresis on a 1% agarose gel, excised, and purified using the Millipore DNA Gel Extraction Kit (Millipore Cat# LSKGEL050). DNA was further purified with QIAquick PCR Purification Columns (Qiagen Cat# 28106). Libraries were prepared from purified mononucleosomal DNA following the protocol in (Gualdrini et al., 2022). Sequencing was performed as 51-bp paired-end reads at high depth using an Illumina platform.