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Loss of non-CpG methylation in inhibitory neurons impairs neural function through a mechanism that partially overlaps with Rett syndrome

ABSTRACT: Whole striatum were dissected from wild-type, as well as in mice where Dnmt3a or MeCP2 was conditional deleted in inhibitory neurons. Inhibitory neuronal nuclei were also conditionally tagged with GFP in all mice. Tissues were snap-frozen in liquid nitrogen until further analysis. Inhibitory neurons were sorted from whole striatum using anti-GFP-488 and anti-NeuN-647 signals and snap frozen until further analysis. Total nuclear RNA was purified from each sample using the Single Cell RNA Purification Kit from Norgen Biotek according to manufactures instructions and stored at -80C until library preparation at the Genomic and RNA Profiling Core at Baylor College of Medicine. NuGEN Ovation RNA-Seq v2 & Rubicon ThruPlex DNA-Seq kits were utilized for library preparation. A paired-end 100 cycle run was used to sequence the flowcell on a HiSeq 2500 Sequencing System.

ORGANISM(S): Mus musculus

PROVIDER: GSE123941 | GEO | 2020/03/16

REPOSITORIES: GEO

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Loss of non-CpG methylation in inhibitory neurons impairs neural function through a mechanism that partially overlaps with Rett syndrome

Project description:Whole striatum were dissected from wild-type, as well as in mice where Dnmt3a or MeCP2 was conditional deleted in inhibitory neurons. Inhibitory neuronal nuclei were also conditionally tagged with GFP in all mice. Tissues were snap-frozen in liquid nitrogen until further analysis. Inhibitory neurons were sorted from whole striatum using anti-GFP-488 and anti-NeuN-647 signals and snap frozen until further analysis. Total DNA was purified from each sample using the DNeasy Blood & Tissue Kit from Qiagen according to manufactures instructions and stored at -20C until library preparation. DNA methylome libraries were generated using a modified snmC-seq method adapted for bulk DNA samples. Paired-end 150 cycle runs was used to sequence the libraries on a Illumina HiSeq 4000 Sequencing System.

2020-03-16 | GSE124009 | GEO

MNase-Seq for budding yeast Saccharomyces cerevisiae

Project description:Libraries were prepared by using two methods. The one is ThruPlex kit from Rubicon Genomics, the other is TCS-ligation based method described in our paper.

2018-02-16 | GSE110666 | GEO

Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: ChIP-seq

Project description:CTCF is a highly conserved and ubiquitously expressed protein involved in several fundamental processes such as fine-tuning gene expression, imprinting, X-chromosome inactivation and 3D chromatin organisation. To understand the impact of differences in the concentration of CTCF abundance on these processes, we exploit a CTCF hemizygous mouse model with a stable reduction in the concentration of this protein. We derived twelve independent primary lines of mouse embryonic fibroblasts (MEFs) from six wildtype and six CTCF-hemizygous mouse E13.5 embryos. MEFs were fixed in DMEM containing 1% fresh formaldehyde and incubated at room temperature for 10 min, quenched with 250mM glycine for 10 min, and washed twice with ice cold PBS, before being flash-frozen at -80°C. Cross-linked cells were lysed and sonicated on a Bioruptor Plus (Diagenode) sonicator to fragment chromatin to an average length of 300bp. 10 ug of the following antibodies were used for immunoprecipitation: CTCF (rabbit polyclonal, Merk Millipore 07-729, lot 2517762); H3K4me3 (mouse monoclonal IgG clone CMA304, Merck Millipore 05-1339, lot 2603814); H3K27ac (rabbit polyclonal IgG, Abcam 4729, lot GR244014-1). Immunoprecipitated DNA or 50 ng of input DNA was used for library preparation using the ThruPLEX DNA-Seq library preparation protocol (Rubicon Genomics, UK). Library fragment size was determined using a 2100 Bioanalyzer (Agilent). Libraries were quantified by qPCR (Kapa Biosystems). Pooled libraries were sequenced on a HiSeq4000 (Illumina) according to manufacturer’s instructions using single-end 50 bp reads. On the same MEF lines we have performed RNAseq and HiC (see related accession numbers).

2018-07-29 | E-MTAB-6261 | biostudies-arrayexpress

T cells facilitate brain metastasis of breast cancer.

Project description:Three breast cancer cell lines were co-cultured with T cells isolated from normal donors, in 3: 1 ratio. As controls, the breast cancer cells were cultured without T cells for three days. All cell cultures were washed three times with PBS to remove T cells before they were scraped and collected in 1.5 mL Eppendorf tubes. Three additional washing steps with PBS were applied. After removing the supernatant, the cell pellets were immediately snap-frozen on dry-ice and stored in -80 °C until the time of preparation. After thawing the samples, they were prepared and measured on nano LC.

2018-01-24 | PXD006750 | Pride

Loss of Capicua alters early T cell development and predisposes mice to T cell lymphoblastic leukemia/lymphoma

Project description:Thymic lymphomas were dissected from Cic adult knockout mice or wildtype mice transplanted with Cic adult knockout progenitors. Tissues were snap-frozen in liquid nitrogen until further analysis. RNAs were purified using the AllPrep DNA/RNA mini kit (Qiagen).

2018-01-18 | GSE107915 | GEO

Combined double knockout of Ezh2 and G9a leads to expression change in murine retinal ganglion cells

Project description:Purpose: The goal of this study is to evaluate H3K27me3 histone marks in retinal ganglion cells in Ezh2 (Math5Cre, Ezh2-/-) and combined G9a-Ezh2 (Math5Cre; G9a+/-Ezh2-/-) knockout mutant mice at P1. Both Ezh2 and G9a are major epigenetic silencing machineries. Ezh2 mediates the trimethylation of lysine 27 on histone 3. Emerging evidences show an interdependence between Ezh2 and G9a to facilitate K27 trimethylation. Methods: P1 retinal ganglion cells were collected and chromatin immunoprecipitation was performed with EZ- CHIP kit from Millipore. Library preparation was done with Rubicon Genomics Thruplex DNAseq. Results: Here, we find a pool of common K27me3 target genes of Ezh2 and G9a in double knockout that leads to dysfunction in double mutant RGCs. Conclusion: This study provides the first Chip-seq analysis of K27me3 in murine RGCs. Our data supports that an interaction between Ezh2 and G9a is also evident in murine retinal ganglion cells. This work should be a framework for further study of retinal ganglion cells and retinal diseases.

2022-07-29 | GSE118011 | GEO

human pituitary and prolactinoma

Project description:Analysis the different gene expression of prolactinomas and normal pituitary tissues. Prolactinoma is a benign pituitary tumor that produces excessive prolactin resulting in hyperprolactinemia and another disease.Tissues were microdissected and removed using the surgical microscope, rinsed in sterile saline, snap-frozen in liquid nitrogen, and stored (-80 ℃) until analysis. Results provide insight into understanding the difference of gene expression in prolacinomas.

2018-09-03 | GSE119063 | GEO

Discovery of a periosteal stem cell mediating intramembranous bone formation and fracture healing (single cell RNA-Seq)

Project description:CTSK-mGFP positive cells from Day 6 old mouse femurs were sorted as single cells into 384 well plates pre-loaded with unique barcoded RT-primers. After sorting, cells were snap frozen on dry ice before being submitted to the New York Genome Center (NYGC) for cDNA synthesis and library preparation. The FACS profile for all the sored cells were collected to co-relate with gene expression.

2018-08-13 | GSE106236 | GEO

Truncation of Pik3r1 Causes Severe Insulin Resistance Uncoupled From Obesity and Dyslipidaemia by Increased Energy Expenditure

Project description:Both PIK3R1(Y657X) mutant and wild-type C57Bl/6J mice were maintained on chow until 16 weeks old, at which time they were fasted for 16 hours and then refed chow for 6 hours. The tissues were harvested and snap-frozen at three time points: chow diet before overnight fast (ad libitum chow, ALC), after 16 hour fast (overnight fast, ONF), and after 6 hour refeeding (refed 6 hours, RF6).

2020-06-30 | GSE153431 | GEO

Analysis of the proteome of individual equine cumulus complexes after maturation in-vitro and in-vivo

Project description:Maturation of oocytes under in-vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in-vivo. Oocytes are closely coupled to their cumulus complex (COC) with a bidirectional exchange of metabolites. Therefore, elucidation of aberrations in cumulus metabolism in-vitro is crucial for a better mimicking of physiological maturation conditions. The aim of this study was the analysis of the equine cumulus cells proteome of single cumulus complexes of metaphase II oocytes matured either under in-vivo (n=8) or in-vitro (n=7) conditions. For in-vivo COC collection mares were slaughtered 30 hours after injection and cumulus complexes from the dominant follicle were harvested for analysis. For in-vitro maturation COCs were recovered from mares out of oestrous and matured for 30 hours in-vitro. COCs were separated in cumulus complexes and oocytes, and only cumulus of successfully matured oocytes was analyzed for this study. All cumulus samples were washed, snap frozen and stored in liquid nitrogen until preparation for analysis.

2019-11-12 | PXD011086 | Pride

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