GSM8804282: A549_null_nucleocapsid_rep2_merged; Homo sapiens; MNase-Seq
Cells were crosslinked with 1% v/v formaldehyde (Sigma-Aldrich) and incubated for 10 minutes at room temperature while rocking. Excess formaldehyde was quenched with 125mM glycine and cells were washed twice in PBS. Cells were resuspended in nuclei isolation buffer (10 mM HEPES at pH 7.8, 2 mM MgOAc2, 0.3 M sucrose, 1 mM CaCl2 and 1% Triton-X) and pelleted by centrifugation at 1000 × g for 10 min at 4 °C. Nuclei were washed twice in nuclei isolation buffer and nuclei purity was confirmed by DAPI staining.  Each condition was processed in biological replicates. For each reaction, 2.5 × 10⁶ nuclei were resuspended in 500 µL of nuclei isolation buffer and digested with Micrococcal Nuclease (MNase) (Worthington) under light (5U) or heavy (200U) conditions for 10 minutes at 37°C. Digestion was stopped with 50 mM EDTA. Protein-DNA crosslinks were reversed by treating MNase-digested nuclei with 0.2 mg/mL proteinase K and 1% SDS, followed by overnight incubation at 65°C. DNA was purified using a guanidinium-isopropanol and ethanol on a silica column. Mononucleosomal and sub-mononucleosomal DNA (<200 bp) was isolated from a 2% agarose gel and purified by glycogen precipitation. MNase sequencing libraries were prepared using the Accel-NGS®2S Plus DNA Library Kit (Swift) for Illumina®, with a minimum of 10 ng input DNA per digestion. Libraries were indexed using the Accel-NGS 2S Unique Dual Indexing Kit, normalized with Normalase, and quality-checked via D1000 Tapestation. Library molar concentrations were determined via KAPA qPCR and size-corrected using Tapestation data. We used a previously validated Roche NimbleGen SeqCap EZ Library (Sexton et al. 2016; Druliner et al. 2016) to capture 2kb regions flanking the transcription start site (TSS) of all human genes. TSS sequences were repeat masked with human COT DNA, and library adapters were blocked using universal oligos. Biotinylated capture oligos were hybridized to input libraries for 48 hours and purified with streptavidin beads. Enriched sequences were amplified via 15-cycle PCR using TruSeq primers. Final libraries were assessed via Tapestation, and molar concentrations were determined using KAPA qPCR. Paired-end 50 bp sequencing was performed on an Illumina NovaSeq 6000 at the FSU College of Medicine.