Exploring the spatiotemporal genetic heterogeneity in metastatic lung adenocarcinoma using a nuclei flow-sorting approach
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PROVIDER: PRJNA490199 | ENA |
REPOSITORIES: ENA
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Project description:Variable tumor cellularity can limit sensitivity and precision in comparative genomics, because differences in tumor content can result in misclassifying truncal mutations as region-specific private mutations in stroma-rich regions, especially when studying tissue specimens of mediocre tumor cellularity such as LUADs. To address this issue, we refined a nuclei flow-sorting approach by sorting nuclei based on ploidy and the LUAD lineage marker thyroid transcription factor 1 (TTF-1) and applied this method to investigate genome-wide somatic copy number aberrations (SCNA) and mutations of 409 cancer genes in 39 tumor populations obtained from 16 primary tumors and 21 matched metastases. This approach increased the mean tumor purity from 54% (range: 7-89%) of unsorted material to 92% (range: 79-99%) after sorting. Despite this rise in tumor purity, we detected limited genetic heterogeneity between primary tumors and their metastases. In fact, 88% of SCNAs and 80% of mutations were propagated from primary tumors to metastases and low allele frequency mutations accounted for much of the mutational heterogeneity. Even though the presence of SCNAs indicated a history of chromosomal instability (CIN) in all tumors, metastases did not have more SCNAs than primary tumors. Moreover, tumors with biallelic TP53 or ATM mutations had high numbers of SCNAs, yet they were associated with a low interlesional genetic heterogeneity. The results of our study thus provide evidence that most macroevolutionary events occur in primary tumors before metastatic dissemination and advocate for a limited degree of CIN over time and space in this cohort of LUADs. Sampling of primary tumors thus may suffice to detect most mutations and SCNAs. In addition, metastases but not primary tumors had seeded additional metastases in three of four patients; this provides a genomic rational for surgical treatment of such oligometastatic LUADs.
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Project description:Chromatin structure plays a pivotal role in facilitating proper control of gene expression. The ability of transcription factors (TF) to bind cis-elements is often associated with accessible chromatin regions. Therefore, identification of these accessible regions throughout plant genomes is important to understanding the relationship between TF binding, chromatin status and the regulation of gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a recently developed technique used to map open chromatin zones in animal genomes. However, in plants, the existence of cell walls, subcellular organelles and the lack of stable cell lines have prevented routine application of this technique. Here, we describe an assay combining ATAC-seq with Fluorescence-Activated Nuclei Sorting (FANS) to identify and map open chromatin and TF-binding sites in plant genomes. FANS-ATAC-seq compares favorably with published DNaseI sequencing (DNase-seq) results and it only requires 500-50,000 nuclei for accurate identification of open chromatin states.
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Project description:We report RNA-seq of single nuclei isolated from the adult C57BL/6 male mouse Hippocampus region. Majority of the nuclei were isolated from 12 weeks old mice (4 different animal), with an additional set of nuclei from 3 months and 2 years old animals. In addition a set of GFP labeled nuclei driven by a VGAT promoter . Microdissections of dentate gyrus, CA1 and CA2/3 regions of the Hippocampus were placed into ice-cold RNA-later for fixation and stored at 4âc overnight, then stored in -80âc. Nuclei were isolated by sucrose gradient centrifugation and kept on ice until sorting using Fluorescence Activated Cell Sorting (FACS) into 96 well plates containing RNA lysis buffer. Single nucleus RNA was first purified then derived cDNA libraries were generated following a modified Smart-seq2 protocol. For VGAT nuclei: high titer AAV1/2 of pAAV-EF1a-DIO-EYFP-KASH-WPRE-hGH-polyA was injected into dorsal and/or ventral Hippocampus, animals were sacrificed two weeks after injections, and GFP labeled nuclei were sorted into plates and processed as described above.
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