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Exploring the spatiotemporal genetic heterogeneity in metastatic lung adenocarcinoma using a nuclei flow-sorting approach

ABSTRACT: Exploring the spatiotemporal genetic heterogeneity in metastatic lung adenocarcinoma using a nuclei flow-sorting approach

PROVIDER: PRJNA490199 | ENA |

REPOSITORIES: ENA

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Exploring the spatiotemporal genetic heterogeneity in metastatic lung adenocarcinoma using a nuclei flow-sorting approach

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.

2018-09-11 | GSE119763 | GEO

Project description:Exploring the Spatiotemporal Genetic Heterogeneity in Metastatic Lung Adenocarcinoma using a nuclei flow-sorting approach

| PRJNA486914 | ENA

Single Nucleus DNA Sequencing of Flow Sorted Archived Frozen and Formalin Fixed Paraffin Embedded Solid Tumors

Project description:Archived tissues are a vast resource of annotated clinical samples. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived frozen and FFPE tumor tissues. Our methods, which include isolation and preparation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching frozen and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest.

2024-06-20 | GSE270364 | GEO

Sorting nitrifiers

Project description:Sorting nitrifying microorganism with targeted cell sorting

| PRJEB81149 | ENA

Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

Project description:We have developed a methodology for isolation of high-quality RNA from cells that are fixed, stained and sorted by flow cytometry that allows routine transcriptomic analysis of highly purified cell populations and single cells. This method, essentially developed by modifying existing staining and sorting protocols, involves fixation of cells with glyoxal, an alternative fixative to commonly used formaldehyde, followed by methanol permeabilization, a 2-step primary and secondary antibody staining and sorting by flow cytometry. The advantage of using glyoxal is that it does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. The dataset comprising mRNA seq libraries from unprocessed or fixed and stained human cancer cells demonstrate that RNA recovered from glyoxal-fixed cells does not retain sufficient glyoxal adducts to impair reverse transcription, and also reveal very few differentially expressed genes between the 2 groups. The dataset derived from fixed and stained cells that were sorted into CCNB1 positive or negative fractions show the applicability of this method for sorting highly purified cell fractions as the CCNB1 positive fractions show strong enrichment for G2-phase cells according to the GO analysis.

2020-09-30 | GSE158177 | GEO

Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes

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.

2016-11-30 | GSE85203 | GEO

NKX2-2 based nuclei sorting on human archival pancreas enables the enrichment of islet endocrine populations for single nucleus RNA sequencing

Project description:Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissociation might alter gene expressions. In this work, we cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from human pancreata. We innovated fluorescence-activated nuclei sorting (FANS) based on the positive signal of NKX2-2 antibody to enrich for nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. We observed comparable endocrine cellular composition and cell type signature gene expression between our snRNA-seq libraries and conventional scRNA-seq libraries generated with live cells from freshly isolated human islets. Our work fills a technological gap and helps to unleash archival pancreatic tissue for molecular profiling targeting the endocrine population. We expect that our protocol can be used to enrich nuclei for transcriptomics study from various populations in the pancreas and in different organs/tissues.

2024-04-01 | GSE252614 | GEO

Genome-wide flow sorting CRISPRi screen identify PTGES3 as a novel therapeutic target of AR

Project description:Genome-wide flow sorting CRISPRi screen identify PTGES3 as a novel therapeutic target of AR

| PRJNA746320 | ENA

Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes

Project description:Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes

| PRJNA336549 | ENA

Single nuclei transcriptomic profiling of human astrocytes and oligodendrocytes in Alzheimer’s disease II

Project description:Single nuclei RNA sequencing (snRNA-seq) dataset characterizing nuclei from a putative astrocyte enrichment method: SOX9-positive sorting.

2022-04-01 | GSE167492 | GEO

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