Project description:Using our robust snRNA-seq protocol effectively enhancing nucleus integrity and RNA quality, we comprehensivly characterize the dynamics of cell populations within mouse epididymal and inguinal adipose tissues during obesity.
Project description:Single nucleus RNA sequencing (snRNA-seq), an alternative to single cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying adipose tissue remodeling during obesity. By integrating nuclear RNA-seq data from adipocyte nuclei of varying sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. Specifically, we characterize dysfunctional hypertrophic adipocytes prevalent in visceral adipose tissues during obesity, exhibiting cellular stress, inflammation and impaired metabolic gene expression. Obesity-induced changes in gene expression profiles of adipocyte subpopulations reveal their distinct contributions to adipose tissue pathophysiology. Our study establishes a robust snRNA-seq method, providing novel insights into the mechanisms orchestrating adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.
Project description:We used snRNA-seq to investigate an entire adult mammalian heart of BL6 mice. Whole hearts were harvested from 4 male mice (12 weeks) after cervical dislocation. The hearts were pooled and nuclei isolated using the Nuclei PURE Prep isolation kit (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s protocol. Sequencing was conducted by Genewiz (Leipzig, Germany) on the 10xGenomics system. Single nuclei were captured in droplet emulsions and snRNA-seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3′ Gel Bead and Library V3 Kit. RNA was controlled for sufficient quality on an Agilent 2100 Bioanalyzer system and quantified using a Qubit Fluorometer.
Project description:Spliceosomal snRNA are key components of small nuclear ribonucleoprotein particles (snRNPs), the building blocks of the spliceosome. The biogenesis of snRNPs is a complex process involving multiple cellular and subcellular compartments, the details of which are yet to be described. In short, the snRNA is exported to the cytoplasm as 3‘-end extended precursor (pre-snRNA), where it acquires a heptameric Sm ring. The SMN complex which catalyses this step, recruits Sm proteins and assembles them around the pre-snRNA at the single stranded Sm site. After additional modification, the complex is re-imported into the nucleus where the final maturation step occurs. Our modeling suggests that during the cytoplasmic stage of maturation pre-snRNA assumes a compact secondary structure containing Near Sm site Stem (NSS) which is not compattible with the formation of the Sm ring. To validate our in silico predictions we employed selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) on U2 snRNA in vivo, ex vivo and in vitro, and U4 pre-snRNA in vitro. For the in vivo experiment HeLa cells were incubated for 10 min at 37°C with NAI or DMSO to final concentration 200 mM. RNA was isolated using Trizol (Sigma) and 200 µl chloroform and precipitated with ethanol at -20°C overnight. For the ex vivo experiment, RNA was isolated from HeLa cells after Protease K treatment at room temperature for 45 min. After incubation, RNA was isolated using equilibrated phenol/chloroform/isoamyl alcohol buffered by folding buffer (110 mM HEPES pH 8.0, 110 mM KCl, 11 mM MgCl2) and cleaned on a PD-10 column according to the manufacturer’s instructions. Isolated RNA was treated with 100mM NAI or DMSO for 10 min at 37°C. For the in vitro experiment, U2WT and U4 pre-snRNA were transcribed by T7 polymerase followed by DNase I (30 min at 37 °C) and Proteinase K (30 min at 37°C) treatments. U2 snRNA was purified on 30 kDa Amicon columns, folded for 30 min at 37°C in 57 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. U4 pre-snRNA was purified on Superdex 200 Increase 10/300GL, folded for 30 min at 37°C in 60 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. All prepared RNA samples (in vitro, ex vivo, in vivo) were used for reverse transcription with the gene-specific primer 5’-CGTTCCTGGAGGTACTGCAA for U2 snRNA and 5’- AAAAATTCAGTCTCCG for U4 pre-snRNA. We used SHAPE MaP buffer (50 mM Tris-HCl pH 8.0, 75 mM KCl, 10 mM DTT, 0.5 mM dNTP, 6 mM MnCl2) and SuperScript II (Invitrogen). Amplicons for snRNAs were generated using gene-specific forward and reverse primers. Importantly, the primers include Nextera adaptors required for downstream library construction. PCR reaction products were cleaned using Monarch PCR&DNA Clean-up Kits. Remaining Illumina adaptor sequences were added using the PCR MasterMix and index primers provided in the NexteraXT DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocol. Libraries were quantified using Qubit (Invitrogen) and BioAnalyzer (Agilent). Amplicons were sequenced on a NextSeq 500/550 platform using a 150 cycle mid-output kit. All sequencing data was analyzed using the ShapeMapper 2 analysis pipeline1. The ‘—amplicon’ and ‘—primers’ flags were used, along with sequences of gene-specific handles PCR primers, to ensure primer binding sites are excluded from reactivity calculations. Default read-depth thresholds of 5000x were used. Analysis of statistically significant reactivity differences between ex vivo and in vivo-determined SHAPE reactivities was performed using the DeltaSHAPE automated analysis tool and default settings2. 1. Busan, S. & Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143-148 (2018). 2. Smola, M.J., Rice, G.M., Busan, S., Siegfried, N.A. & Weeks, K.M. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10, 1643-69 (2015).
Project description:In this project, we isolate U1 snRNA associated proteins in Arabidopsis thaliana. We used an antisense oligonucleotide specific for the U1 snRNA and analyzed associated proteins by mass spectrometry. As a control, the same experiments were performed with U2 snRNA- and lacZ-specifc antisense oligonucleotides.
Project description:In this work, we compared different protocols to prepare single-cell suspensions used for scRNAseq and suggest an optimized dissociation protocol for mouse retina, which preserves cell morphology to a higher level leading to an overall increase of gene number per cell. We compared scRNAseq libraries generated with our optimized protocol to publicly available scRNAseq data of mouse retina. We further demonstrate a pipeline to reduce noise in scRNAseq caused by multiplets and ambient RNA.
Project description:We used snRNA-seq to investigate for the first time an entire adult mammalian heart. To avoid potential aberrations due to inbreeding, we relied on an outbred mice strain (Fzt:DU) (Dietl, G.; Langhammer, M.; Renne, U. Model simulations for genetic random drift in the outbred strain Fzt:DU. Arch. Anim. Breed. 2004, 47, 595–604). Whole hearts were harvested from 4 male mice (12 weeks) after cervical dislocation. The hearts were pooled and nuclei isolated using the Nuclei PURE Prep isolation kit (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s protocol. Sequencing was conducted by Genewiz (Leipzig, Germany) on the 10xGenomics system. Single nuclei were captured in droplet emulsions and snRNA-seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3′ Gel Bead and Library V3 Kit. RNA was controlled for sufficient quality on an Agilent 2100 Bioanalyzer system and quantified using a Qubit Fluorometer. For further experimental details as well as computational scripts and results can be obtained from http://doi.org/10.15490/FAIRDOMHUB.1.STUDY.713.1.
Project description:Abstract White adipose tissue (WAT) is a robust energy storage and endocrine organ critical for maintaining metabolic health as we age. Our aim was to identify cell-specific transcriptional aberrations that occur in WAT with aging. We leveraged full-length snRNA-Seq and histology to characterize the cellular landscape of human abdominal subcutaneous WAT in a prospective cohort of 10 younger (≤30 years) and 10 older individuals (≥65 years) balanced for sex and body mass index (BMI). The older group had greater cholesterol, very-low-density lipoprotein, triglycerides, thyroid stimulating hormone, and aspartate transaminase compared to the younger group (p< 0.05). We highlight that aging WAT is associated with adipocyte hypertrophy, increased proportions of lipid-associated macrophages and mast cells, an upregulation of immune responses linked to fibrosis in pre-adipocyte, adipocyte, and vascular populations, and highlight CXCL14 as a biomarker of these processes. We show that older WAT has elevated levels of senescence marker p16 in adipocytes and identify the adipocyte subpopulation driving this senescence profile. We confirm that these transcriptional and phenotypical changes occur without overt fibrosis and in older individuals that have comparable WAT insulin sensitivity to the younger individuals.