GSM8524584: KNF1F2-TET2, replicate 2; Mus musculus; ChIP-Seq
For all organoid 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 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. Chromatin extract was sonicated with QSonica Q800R (pulse: 30 s on/30 s off; sonication time: 20 min; amplitude: 70%) then 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 Dynabeads: FoxA1 (Abcam, ab170933, rabbit monoclonal, 5 µg/ChIP). For in vivo experiments, KN and KNF1F2 tumor-bearing mice were euthanized at 14 weeks post tumor initiation and the rib-cage was dissected to reveal the trachea and heart. Lungs were perfused with cold PBS, removed, and snap frozen in liquid nitrogen. Flash-frozen lungs were minced on ice in 2 ml of ice-cold PBS for 3 – 5 min. Minced lungs were dounced 10 times with a large 7 ml homogenizer (Wheaton) and centrifuged for 5 min at 4°C (300G). Tissue was crosslinked in 2 mM DSG buffer and 1% Formaldehyde as previously described (In vitro organoid ChIP-seq). After fixation, lung samples were resuspended in 2 – 3 ml of TST buffer (Slyper et al. 2020) with protease inhibitors for 5 min. During this time, samples were dounced an additional 5 times in a small 2 ml homogenizer (Wheaton) to extract nuclei. Lysis was quenched with 5 ml of 1X ST buffer (Slyper et al. 2020) plus protease inhibitors. Nuclei were washed in cold PBS and resuspended in 5 – 10 ml cold PBS supplemented with DAPI and protease inhibitors. Nuclei suspension was then sequentially filtered through a 70 μm and 35 μm cell strainer. Nuclei were sorted using BD FACSAria with the 85 μm nozzle for GFP-positive, DAPI-positive nuclei. Samples were sorted into 1 ml of cold PBS with 1% BSA and 10X protease inhibitor. Approximately 3 million nuclei were sorted for histone ChIP-seq and 10 million for TF ChIP-seq experiments.  After sorting, nuclei were pelleted for 10 min at 4°C (500G) then resuspended in Chromatrap Hypotonic and Lysis buffers and sonicated as previously described (In vitro organoid ChIP-seq). Chromatin was immunoprecipitated with antibodies premixed with Protein A Dynabeads: FoxA1 (Abcam, ab170933, rabbit monoclonal, 5 µg/ChIP), TET3 (Sigma, ABE290, rabbit polyclonal, 5 µg/ChIP) and H3K27ac (Active Motif, 39133, rabbit polyclonal, 5 µg/ChIP). ChIP-seq libraries were prepared using the ChIP-Seq with NEBNext Ultra II DNA Library Prep Kit. 10ng of DNA was used as strating material for input and IP samples.