title: GSM8037123: THP-1, 1hr, 200UMNase; Homo sapiens; MNase-Seq
library_construction_protocol: Cells were crosslinked in 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 (10mM HEPES at pH 7.8, 2mM MgOAc2, 0.3M sucrose, 1mM 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 chromatin was digested with either 5U or 200U MNase for 10 minutes at 37°C. MNase reactions were halted with 50mM EDTA. Protein-DNA crosslinks were reversed by treating the MNase-digested nuclei with 0.2 mg/mL proteinase K and 1% sodium dodecyl sulfate, and incubating overnight at 65°C. DNA was isolated using guanidinium-isopropanol and washed with ethanol on a silica column. Mononucleosomal and sub-mononucleosomal DNA was isolated from a 2% agarose gel slice and purified by glycogen precipitation. MNase sequencing libraries were prepared for each replicate using the Accel-NGS®2S Plus DNA Library Kit (Swift) for Illumina® using 10ng of input DNA from each digestion. Libraries were indexed with the Accel-NGS 2S Unique Dual Indexing Kit and normalized with Normalase. We used a previously validated custom designed Roche Nimblegen SeqCap EZ Library SR to capture 2000 bp regions flanking the Transcription Start Site (TSS) for every pol II gene in the human genome(Druliner et al. 2016; Sexton et al. 2016). The TSS sequences were repeat masked using human COT DNA and library adapters were masked with universal blocking oligos. Biotinylated sequence capture oligos were hybridized to input libraries for 48 hours and purified with streptavidin beads. Enriched sequences were amplified in a 15 cycle PCR amplification using TruSeq primers.