title: GSM8911567: 24515X1_GEMM_KN_HED934_input; Mus musculus; ChIP-Seq
library_construction_protocol: For all organoid ChIP-seq experiments, two 24-well plates (approximately 4 – 8 million cells) were collected in Cell Recovery Solution (500 μl per dome). Organoids were submerged in ice for 30 min before centrifugation at 4°C (300G). Cells were 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). After the third wash, organoids were resuspended in 5 ml of 2 mM DSG buffer (1X PBS, 1 mM MgCl2) and rotated at room temperature for 35 min. Formaldehyde was then added to a final concentration of 1% and cells were crosslinked for 10 min. The cross-linking reaction was stopped with the addition of glycine to a final concentration of 125 mM. Cells were washed with cold PBS, then frozen at -80. Cell pellets were thawed on ice for 5 min then lysed in 1 ml of Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40) for all TF experiments or 400 ul of Chromatrap Hypotonic buffer (catalog #100007) for all histone experiments. 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) or 100 μl of Chromatrap Lysis buffer (catalog #100005) for TF and histone ChIPs respectively. All lysis buffers were supplemented with protease inhibitors. Chromatin extract was sonicated with QSonica Q800R (pulse: 30 s on/30 s off; sonication time: 20 min; amplitude: 70%). After sonication, chromatin extract of histone samples was brought up to a volume of 1 ml with RIPA lysis buffer. Chromatin from all samples was immunoprecipitated with antibodies premixed with Protein A/G Dynabeadsper antibody used. For sorted nuclei from GEMMs,  At 14 weeks post-tumor initiation, KN and KNH tumor-bearing mice were euthanized. The rib cage was dissected to expose the trachea and heart, and lungs were perfused with cold PBS, snap-frozen, and minced on ice. Homogenized tissue was centrifuged (300G, 4°C, 5 min) and crosslinked in 2 mM DSG + 1% formaldehyde. After fixation, nuclei were extracted using TST buffer, dounced, and filtered through 70 μm/35 μm strainers. GFP-positive nuclei were sorted using BD FACSAria (85 μm nozzle) into PBS + 1% BSA + protease inhibitors.For ChIP-seq, nuclei (10M for TF ChIP-seq) were pelleted (500G, 4°C, 10 min), lysed, and sonicated (QSonica Q800R, 30s on/off, 20 min, 70%). Chromatin was immunoprecipitated with antibodies premixed with Protein A/G Dynabeads per antibody used. 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).