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: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: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 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: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)
Project description:The chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on the prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to the recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. Genetic ablation of catalytic subunit of the PRC1 complex (RINGA/B) and ChIP-seq analysis of PRC1 and PRC2 components confirmed genome-wide decreases in PRC2 occupancy and H3K27me3 levels at PRC target sites. This activity is restricted to variant PRC1 complexes and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for polycomb domain formation and normal development. Together these observations provide a surprising new PRC1-dependent logic for PRC2 occupancy and polycomb domain formation. RING1A-/-;RING1Bfl/fl ES cells were treated with 800M-BM-5M tamoxifen for 48hours and compared to untreated control cells by ChIP-seq for RING1B, SUZ12, EZH2 and H3K27me3.
