title: GSM8633636: H358_Control_Input_Replicate2; Homo sapiens; ChIP-Seq
library_name: GSM8633636
library_construction_protocol: For cell line ChIP-seq, ~1 x 107 cells were collected per ChIP experiment. For organoid ChIP-seq, ~4 x 106 cells were collected per ChIP experiment. For ChIP-re-ChIP, ~3 x 107 cells were collected per ChIP experiment. For organoid ChIP experiments, organoids were first collected in Cell Recovery Solution and incubated on ice for 30 mins. Organoids were then washed three times in cold PBS. On the second wash, PBS was supplemented with DNase solution (containing a final concentration of 200U/ml DNase [Worthington], 2.5 mM MgCl2, 500 mM CaCl2). 2D adherent cell lines were washed 2X in cold PBS, then cells were scraped and collected and washed once more. Cell/organoid pellets were then resuspended in 5 ml of 2 mM DSG buffer (1X PBS, 1 mM MgCl2) containing 40μL of 0.25 M disuccinimidyl glutarate (DSG) (Thermo Fisher Scientific) and rotated 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) or Protein A Dynabeads (for rabbit antibodies; ThermoFisher Scientific, 10002D): HNF4α (Perseus Proteomics PPH1415), NKX2-1 (Abcam ab133737), FoxA1 (Abcam, ab170933), FoxA2 (CST 8186). 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). At this point, ChIP-re-ChIP samples were subjected to an extra processing step: samples were resuspended in 10mM DTT and incubated with agitation at 37°C for 40 minutes. DTT was diluted by adding 1mL RIPA buffer with PPI, and diluted chromatin was immunoprecipitated for a second time overnight with the reciprocal antibody (either FoxA1 or HNF4α). 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).