Project description:CUT&RUN-seq of H3K4me3, H3K27me3 and H3K27ac in mouse ESCs carrying a homozygous point mutation in the catalytic domain of mll2[Y2602A]. CUT&RUN was performed according to Skene & Henikoff, 2017 and purified DNA was using for library preparation with NEBNext DNA Ultra II kit (NEB E7645S; input: 5 ng of DNA). Libraries were multiplexed and sequenced on a NextSeq500 (Paired-End; read length 40). Each sample is present in 3 biological replicates.
Project description:PolyA RNAseq of mouse ESCs and EpiLCs carrying a homozygous point mutation in the catalytic domain of mll2[Y2602A]. Total RNA was isolated from cell pellets using NEB Monarch Total RNA Miniprep Kit and libraries prepared using NEBNext RNA Ultra II kit (NEB E7770S, input: 350 ng of total RNA). Libraries were multiplexed and sequenced on a NextSeq500 (Paired-End; read length 40). Each sample is present in 3 biological replicates.
Project description:Single-cell proteomics (SCP) reveals heterogeneity and biological insights inaccessible to bulk analysis. Key limitations are cost, sample loss during processing, and accessibility to state-of-the-art instrumentation. We describe a label free SCP methodology in human tissue, combining fluorescence activated cell sorting (FACS), oil-immersion cell handling, mass-spectrometry, and neural-network derived spectral libraries which aims to improve user uptake. We tested this methodology in a skin tumor syndrome, CYLD cutaneous syndrome (CCS), assessing tumor heterogeneity. Using a Bruker timsTOF HT platform we quantified > 4000 proteins, averaging ~700 per cell, through a cost-effective pipeline without specialised liquid handling infrastructure. By utilising pre-existing bioinformatic tools from the scRNA-seq field we implemented a robust analysis methodology, discriminating between macrophages, dendritic cells and tumor keratinocytes, in an unbiased analysis of 419 CCS tumor cells. We confirmed the biological accuracy of cell annotations by cross referencing with each cell's FACS markers. Furthermore, we identified novel CCS tumor biology in a tumor associated macrophage population with a tumor microenvironment remodelling signature. Our findings demonstrate an accessible SCP technology capable of yielding novel biological discoveries in clinical tissue.
Project description:Adipose tissue mass and adiposity change throughout the lifespan. During aging, while visceral adipose tissue (VAT) tends to increase, peripheral subcutaneous adipose tissue (SAT) decreases significantly. Unlike VAT, which is linked to metabolic diseases, SAT has beneficial effects. However, the molecular details behind aging-associated loss of SAT remain unclear. Here we compare scRNA-seq of total SVF of SAT from young and aging mice to identify a novel Aging-dependent Regulatory Cell (ARC) that emerges in SAT of aged mice. Inguinal white adipose tissue (iWAT) was used as a representative SAT; iWAT pads of 2 mice from each age group were subjected to collagenase digestion and treated with a hypotonic buffer to remove red blood cells before subjection to scRNA-seq by 10X Genomics Chromium Single Cell Kit. The findings showed that ARCs express adipogenic markers but lack adipogenic capacity and inhibit differentiation of neighboring adipose precursors.
Project description:The rate-limiting step in glutathione (GSH) synthesis is controlled by glutamate-cysteine ligase catalytic subunit. To investigate the impact of GSH in vivo, we induced a deletion of Gclc using a Gclcf/f Rosa26-CreERT2 mouse model and harvested liver tissue for analysis.
Project description:We performed single-nucleus RNA-seq and single-nucleus methyl-3C seq on subcutaneous adipose tissue (SAT) biopsies from Finnish women who underwent abdominal SAT liposuction at Tilkka Hospital, Helsinki, Finland. The purpose of this study was to understand the epigenomic, 3D topology, and transcriptomic dynamics across the SAT cell-types.
Project description:We performed single-nucleus RNA-seq and single-nucleus methyl-3C seq on subcutaneous adipose tissue (SAT) biopsies from Finnish women who underwent abdominal SAT liposuction at Tilkka Hospital, Helsinki, Finland. The purpose of this study was to understand the epigenomic, 3D topology, and transcriptomic dynamics across the SAT cell-types.
Project description:The use of single cell RNA sequencing (scRNA-seq) remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Here, we investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. We found that LP-FACS readily outperforms conventional FACS for isolation of structurally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FAC-isolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties.
Project description:Three Vsx2-GFP mouse retinas were dissected, dissociated and FACS sorted, and single cell RNA-seq libraries were generated for 288 single cells and 3 bulk libraries using Smart-seq2 (~10,000 cells each)
