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Organelle isolation Single cell analysis Tissue analysisNuclei Isolation Methods on Frozen Clotted Blood Samples
It is common practice for laboratories to discard clotted blood or freeze it for future DNA extraction after extracting serum from a serum-separating tube. If freezing for DNA extraction, the blood clot is not usually cryopreserved, which leads to cell membrane fragility. In this protocol, we describe steps to isolate high-quality nuclei from leukocytes derived from whole blood samples frozen without a cryoprotective medium. Nuclei isolated from this protocol were able to undergo ATAC (assay for transposase-accessible chromatin) sequencing to obtain chromatin accessibility data. We successfully characterized and isolated B cells and T cells from leukocytes isolated from previously frozen blood clot using Miltenyi’s gentleMACS Octo Dissociator coupled with flow sorting. Nuclei showed round, intact nuclear envelopes suitable for downstream applications, including bulk sequencing of nuclei or single-cell nuclei sequencing. We validated this protocol by performing bulk ATAC-seq.
Optimized Method for High-Quality Isolation of Single-Nuclei From Mosquito Fat Body for RNA Sequencing
Single-cell and single-nucleus RNA sequencing are revolutionizing our understanding of cellular biology. The identification of molecular markers, single-cell transcriptomic profiling, and differential gene expression at the cellular level has revealed key functional differences between cells within the same tissue. However, tissue dissociation remains challenging for non-model organisms and for tissues with unique biochemical properties. For example, the mosquito fat body, which serves functions analogous to mammalian adipose and liver tissues, consists of trophocytes—large, adipocyte-like cells whose cytoplasm is filled with lipid droplets. Conventional enzymatic dissociation methods are often too harsh for these fragile cells, and their high lipid content can interfere with reagents required for single-cell transcriptomic analysis. Single-nucleus RNA sequencing (snRNA-seq) offers an alternative strategy when intact cells with high-quality RNA cannot be obtained by enzymatic or mechanical dissociation. Here, we present an optimized reproducible methodology for nuclei isolation from the fat body of Anopheles gambiae mosquitoes, enabling high-quality snRNA-seq. Our approach involves tissue fixation and lipid removal, followed by cell lysis and nuclei purification using a sucrose cushion. We validated this protocol on both sugar-fed and blood-fed samples, established quality metrics to remove potential ambient RNA contamination, and demonstrated that snRNA-seq using this method yields high-quality sequencing results.
Preparation of Chromatin Fragments From Human Cells for Cryo-EM Analysis
Eukaryotic genomic DNA is packaged into chromatin, which plays a critical role in regulating gene expression by dynamically modulating its higher-order structure. While in vitro reconstitution approaches have offered valuable insights into chromatin organization, they often fail to fully capture the native structural context found within cells. To overcome this limitation, we present a protocol for isolating native chromatin fragments from human cells for cryo-electron microscopy (cryo-EM) analysis. In this method, chromatin from formaldehyde-crosslinked human HeLa S3 nuclei is digested with micrococcal nuclease (MNase) to generate mono- and poly-nucleosome fragments. These fragments are subsequently fractionated by sucrose-gradient ultracentrifugation and prepared for cryo-EM. The resulting chromatin fragments retain native-like nucleosome–nucleosome interactions, facilitating structural analyses of chromatin organization under near-physiological conditions.
Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing
Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.
Fractionation and Extraction of Crude Nuclear Proteins From Arabidopsis Seedlings
The plant nucleus is an important subcellular organelle that contains the genome, ribosomal RNA, and regulatory proteins, and performs a central role in the functioning and metabolism of the cell. Fractionation of intact nuclei is a crucial process to elucidate the function of nuclear proteins. Here, we present a simple method for the fractionation of crude nuclei and extraction of nuclear proteins, based on previously established methods. This protocol provides an easy and quick method to isolate crude nuclei and extract nuclear proteins from Arabidopsis seedlings, which is useful for the research on the nuclear proteins, without requirement for high-purity nuclei.
Graphic abstract:
Schematic procedure for the isolation of crude nuclei and extraction of nuclear proteins from Arabidopsis seedlings.
ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach
NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
Nuclei Isolation from Nematode Ascaris
Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
An Efficient Procedure for Protoplast Isolation from Mesophyll Cells and Nuclear Fractionation in Rice
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