Protocol to extract and sort GFP-tagged nuclei from lung adenocarcinoma tissue in genetically engineered mouse models

Here, we present a protocol to isolate GFP-positive nuclei from whole lung tumor tissue from a genetically engineered mouse model (GEMM) for downstream analysis. First, we outline the procedure for the isolation of fixed GFP-positive nuclei for utilization in chromatin immunoprecipitation followed by sequencing (ChIP-seq) assay. We also describe the sorting of unfixed nuclei for whole-genome methylation analysis. This nuclei collection methodology can be adapted for the retrieval of other fluorescently tagged nuclei and/or for use in other downstream analysis. For complete details on the use and execution of this protocol, please refer to Gillis et al. 1 • Protocol for the isolation of nuclei from fresh and frozen murine lung tumors • Preparation of cells for fluorescence-activated nuclei sorting (FANS) • Sorted nuclei for in vivo chromatin immunoprecipitation sequencing (ChIP-seq) • Sorted nuclei for in vivo methylation sequencing Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to isolate GFP-positive nuclei from whole lung tumor tissue from a genetically engineered mouse model (GEMM) for downstream analysis. First, we outline the procedure for the isolation of fixed GFP-positive nuclei for utilization in chromatin immunoprecipitation followed by sequencing (ChIP-seq) assay. We also describe the sorting of unfixed nuclei for whole-genome methylation analysis. This nuclei collection methodology can be adapted for the retrieval of other fluorescently tagged nuclei and/or for use in other downstream analysis.