Cell Biology

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.

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.

Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as 100 human or 1,000 Drosophila cells. The method employs tagmentation to fragment the chromatin with concomitant addition of sequencing adaptors. The method generates datasets with high signal to noise ratio and can be subjected to standard tools for ChIP-Seq analysis

This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts.

Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA analyses, or preparation of nuclear extracts (Kang et al., 2016; Wang et al., 2016).

Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. The extent of association with chromatin changes rapidly in response to stresses, such as immune activation, oxidative stress, or viral infection, resulting in downstream effects on chromatin conformation and transcription of target genes. To elucidate changes in the composition of proteins associated with chromatin under different conditions, we adapted existing protocols to isolate nuclei and fractionate cellular chromatin using a gradient of salt concentrations. The presence of specific proteins in different salt fractions can be assessed by Western blotting or mass spectrometry, providing insight into the degree to which they are associated with chromatin.

Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. This protocol, adapted from Zhang et al. (2011), describes a procedure for the isolation of rice protoplasts from green tissue and shows an efficient and rapid method for isolation of nuclei form these protoplasts which are commonly used in a variety of experimental procedures including the isolation of high-molecular-weight DNA (Watson and Thompson, 1986), in vitro DNA synthesis (Roman, 1980), isolation of labeled transcripts for differential screening of cDNA libraries (Somssich et al., 1989), preparation of nuclear extracts for in vitro transcription systems (Roberts and Okita, 1991), isolation of nuclear proteins (Harrison et al., 1992) and studies of protein targeting to the nucleus (Hicks and Raikhel, 1993).

This protocol describes how to extract nuclear protein from mouse lungs, including tissue preparation, stepwise lysis of cells and centrifugal isolation of nuclear protein fraction. This is an efficient method to get comparable nuclear protein extracts from lung tissues.

Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample handling this method has high recovery and good reproducibility. As an example, in this protocol we describe the isolation of cell nuclei from adult cerebral cortex, which are subsequently immunostained with antibodies against NeuN (a general neuronal marker) and EGR1 (an early response gene expressed by functionally active neurons), and subjected to flow cytometric gating and analysis. Nevertheless, the protocol can also be applied to other neural tissues from adult and embryonic brain.

Micrococcal nuclease (MNase) is able to produce double-strand breaks within nucleosome linker regions. The efficiency of MNase digestion depends on the degree of chromatin compaction, being more easily digested the regions of less compacted chromatin. The MNase protocol described here can be used to asses changes in the chromatin structure of nuclei extracted from Arabidopsis seedlings.