Name: GSM7008795
Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: ChIP was performed with an adapted disuccinimidyl glutarate (DSG) and formaldehyde chromatin crosslinking protocol (Wang 2022, Tian 2012, Singh 2019). 2-5x106 cells were collected in PBS, fixed with 2mM DSG reagent (Thermo Fisher Scientific) for 45min rotating at room temperature, washed, fixed with 1% formaldehyde (Thermo Fisher Scientific) for 10min rotating at room temperature, and quenched with 125mM glycine rotating for 5min at room temperature. Fixed cells were lysed in Buffer 1 (50mM HEPES-KOH, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, freshly supplemented protease and phosphatase inhibitors) by rotation for 10min at 4ºC, pelleted for 5min at 3000rpm at 4ºC, lysed in Buffer 2 (10mM Tris-HCl pH8, 200mM NaCl, 1mM EDTA pH8, 0.5mM EGTA, freshly supplemented protease and phosphatase inhibitors) by rotation for 10min at 4ºC, pelleted for 5min at 3000rpm at 4ºC, and resuspended in Buffer 3 (10mM Tris-HCl pH8, 100mM NaCl, 1mM EDTA pH8, 0.5mM EGTA, 0.1% NaDOC, 0.5% N-laurylsarcosine, freshly supplemented protease and phosphatase inhibitors) for sonication. Cells were sonicated on High for 30min with 30sec on/off in a water bath sonicator maintained at 4ºC (Diagenode). 10% Triton X-100 was added to sonicated lysates and spun at 11,000rpm for 10min at 4ºC to pellet debris; 5% input and the appropriate number of aliquots were taken from sonicated lysate. Following overnight antibody incubation and incubation with BSA-blocked Dynabeads (beads washed in RIPA buffer were incubated with 0.1ug/uL BSA for 30min followed by RIPA wash) for 1h at room temperature, beads were sequentially washed in low salt (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA pH8, 1% Triton X-100, 0.1% SDS), high salt (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA pH8, 1% Triton X-100, 0.1% SDS), and LiCl (250mM LiCl, 10mM Tris-HCl pH8, 1mM EDTA pH8, 1% NP-40, 1% NaDOC) buffers. DNA was eluted with Elution buffer (1% SDS, 100mM NaHCO3) and agitation for 15min at 42ºC. ChIP and input DNA was reverse crosslinked with RNaseA (0.2mg/mL) and NaCl (20mM) for 2h at 65ºC and Proteinase K (0.4mg/mL) for 1h at 50ºC before processing using Qiagen PCR Clean Up Kit per kit protocol ChIP-seq libraries (Illumina) were prepared according to manufacturer's recommendations and sequenced at the UNC High Throughput Genomic Sequencing facility using paired-end 50bp reads