GSM8524618: KN-H3K27ac, replicate HiChIP 2; Mus musculus; OTHER
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 (1X PBS, 1 mM MgCl2) for 35min, then formaldehyde was added to final concentration of 1% and incubated for 10min. The cross-linking reaction was stopped with the addition of glycine to a final concentration of 125 mM. 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 10 million nuclei were sorted for HiChIP experiments. HiChIP protocol based on Mumbach et al., 2016, Nature Methods. Cross-linked chromatin was digested with the MboI restriction enzyme followed by end-repair with dNTPs including biotin labeled dATP, ligation using T4 DNA ligase, and sonication to obtain 1kb chromatin fragments using Qsonica (Q800). To enrich for chromatin interactions occurring at active regulatory elements, an anti-H3K27ac antibody (Abcam, ab4729, rabbit polyclonal, 7.5ug/HiChIP) was used for DNA fragment capture. Streptavidin magnetic beads were used to pull down ligated DNA fragments and HiChIP libraries were prepared using Illumina Tagment DNA Enzyme and Buffer Kit. Sequencing was performed with the Illumina Nextera Library preparation.