title: GSM8690804: 22437X13_ASPC1_sgP2D_Input_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-seq, 10 million cells were collected per replicate. Cells were washed 3X in cold PBS, then resuspended in 10 ml of 2 mM DSG buffer (1X PBS, 1 mM MgCl2) containing 80μL of 0.25 M disuccinimidyl glutarate (DSG) (Thermo Fisher Scientific) and incubated at room temperature for 35 min. Methanol-free formaldehyde was then added to a final concentration of 1% and cells were crosslinked for 10 min. The cross-linking reaction was quenched with the addition of glycine to a final concentration of 125mM. Cells were washed with cold PBS, then the cell pellet was snap frozen in liquid nitrogen and stored at -80°C. Cell pellets were thawed on ice for 5 minutes then lysed in 1 mL of Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40) with Pierce Protease Inhibitor (PPI, ThermoFisher Scientific, A32959). Samples were centrifuged at 4°C (1000G) then resuspended in 1mL of RIPA lysis buffer (1X PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with PPI. Chromatin was sonicated with a QSonica Q800R (pulse: 30 s on/30 s off; sonication time: 20 minutes; amplitude: 70%). After sonication, samples were centrifuged at 17,000 x g for 15 minutes and an input was collected from each sample of sheared chromatin. Chromatin from each sample was then immunoprecipitated overnight with 5ug of the following antibodies (per sample) premixed with Protein G Dynabeads (for mouse antibodies; ThermoFisher Scientific, 10004D): HNF4α (Perseus Proteomics PP-H1415) and HNF4α-P1 (Perseus Proteomics PP-K9218) The next day, bound chromatin was washed 5 times with LiCl Wash Buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate). For all samples, crosslinks were reversed by incubating samples in IP Elution Buffer (1% SDS, 0.1% NaHCO3) overnight at 65°C. ChIP DNA was purified using Zymo ChIP DNA Clean and Concentrator Kit (Zymo, D5205). Library preparation was performed using the ChIP-seq with NEBNext DNA Ultra II library prep kit using Unique Molecular Indexes (UMIs). Sequencing was performed using the Illumina NovaSeq 6000 (150 x 150 bp paired-end sequencing, 25 million reads per sample).