Single-nuclei sequencing of Parvalbumin interneurons in mouse visual cortex after optoTrkB activation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE142797 | GEO | 2023/01/06
REPOSITORIES: GEO
Similar Datasets
Project description:Genome-wide gene expression was obtained in three auditory brainstem nuclei (defined below), at two different ages in mice, postnatal day (P)3 and P14. The primary aim was to identify genes which are differentially expressed between the medial nucleus of the trapezoid body (MNTB) and the superior olive (LSO), at both age groups. Tissue samples from the ventral cochlear nucleus (VCN), the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) were collected from 200 μm thick fresh transverse mouse brainstem slices prepared with a vibratome tissue slicer. Individual brainstem nuclei were dissected out with fine forceps under optical control with a binocular. For each nucleus (VCN, MNTB, and LSO), the tissue from six male C57Bl6 mice at postnatal day (P) 3, and from three male C57Bl6 mice at P14 was pooled. The RNA was isolated with the RNeasy Micro kit (Qiagen); RNA samples were amplified using a WT ovation Pico RNA amplification (Nugen, CA, USA). The cDNA was hybridized onto mouse Affymetrix microarrays 430 2.0 (Affymetrix, Santa Clara, USA).
2013-05-01 | E-GEOD-46500 | biostudies-arrayexpress
Project description:The transcriptomics and BCR-rep data was meassured at the single-cell level from the longitudinal samples obtained from an individual following Boostrix vaccination. The donor had no known or suspected exposure to pertussis infection nor vaccination in the past 10 years. EDTA-blood samples for single-cell sequencing were collected at baseline and days 5 (d5), d7, d10 and d14 post-vaccination. At baseline, first 10,000 total CD19+ B cells were sorted, and after adjusting the sorting gate to exclusively sort PB/PCs, another 1,201 CD19+CD38++CD24+CD27+ PB/PCs were sorted. For subsequent visits, all available PB/PCs were sorted (11,051 cells). In total, we sorted 22,252 cells. Nearly 22,000 B cells enriched in PB/PCs from different time-points were processed into single cells in a Chromium Controller (10X Genomics). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 3’ v3 protocol (10X Genomics) per manufacturer’s recommendations. After amplification of the cDNA, a 5 ́gene expression library and paired heavy and light chain library were generated from cDNA of the same cell using Chromium Single Cell VDJ reagent kit (scBCR-rep library; v1.1chemistry, 10X Genomics). After library preparation, quality control was performed using a bioanalyzer (Agilent 2100 Bioanalyzer; Agilent Technologies). The libraries were sequenced in the NovaSeq6000 sequencer (Illumina) using the v1.0 sequencing reagent kit (read length: 2 x 150bp).
2021-10-08 | GSE185427 | GEO
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.
2020-03-16 | E-MTAB-8848 | biostudies-arrayexpress
Project description:Different lesion types were microdissected out from snap-frozen white matter tissue. Brain nuclei were fluorescence-activated nuclear sorted. Gel beads-in-emulsion was generated, barcoded, reverse transcribed followed by library construction and sequencing.
2024-06-21 | GSE231585 | GEO
Project description:Purpose: Alteration in light dark cycle leads to changes in daily behavior in mammals. We have previously demonstrated that different light:dark cycle lengths result in dynamic changes in DNA methylation within the suprachiasmatic nuclei (SCN) that globally alter transcription of both clock genes and non-clock genes. The goal of this study was to re-examine DNA methylation in the SCN separately for dorsal and ventral regions. Method: Six-week-old wild-type mice (C57BL/6Cr1) were entrained to light:dark cycles either 22, 24, or 26 hours in length (T22, T24, or T26) for six weeks. After 6 weeks, we recorded wheel-running behavior in constant darkness. After 3 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at the circadian time CT4 and then sliced immediately using vibratome, one coronal SCN slice of ~250μM was collected from the middle part of the SCN. Using ophthalmic knife vSCN and dSCN sub-regions were separated. To get enough DNA, a pool of vSCN and dSCN was prepared twice from 5 mice from each T-cycle. Genomic DNA was extracted from SCN slices by overnight Proteinase K digestion, followed by phenol-chloroform and ethanol precipitation. After RNase treatment, genomic DNA was sonicated to generate small fragments (average of 300bp), using a Covaris sonicator (BioRuptor). DNA immunoprecipitation was performed using Epiquik™ MeDIP Ultra Kit (EPIGENTEK™), following manufacturer's instructions. The methylated enriched DNA was then used for library preparation using Accel-NGS™ DNA Library Kits (Swift Biosciences), following manufacturer's instructions. Quality check, library preparation and sequencing were performed by Fasteris (CH). Results: 13-21 x 10^6 reads were obtained from each meDIP library. Quality control was performed on the fasta raw sequence data using FastQC.MeDIP-seq data were aligned to the mm9 mouse genome build using the Burrows-Wheeler Alignment tool (BWA) (Li and Durbin, 2009). Mapped reads were than analyzed using the MEDIPS package (Lienhard et al., 2014). Methylation score was calculated for each region of 500bp across the genome. CpG density-dependent normalization was incorporated. Pairwise correlation analysis of the genome wide coverage profile between MEDIP replicate samples was performed using the MEDIPS.correlation function. Pearson correlation coefficient r varied between 0.85 and 0.92, indicating good reproducibility of MEDIP-seq signal. Differential methylation analysis was performed using MEDIPS in 500bp widows excluding regions with less than 8 unique mapped reads. DMR were than identified by filtering for a widows associated with a p ≤0.001. DMR were than mapped using the web based chip-seq tool “ChIPseek” (Chen et al., 2014). Different sets of genes showed methylation changes in dorsal vs. ventral regions. Interestingly, far greater changes were observed in vSCN than in dSCN.
2016-10-28 | GSE89255 | GEO
Project description:Different lesion types were microdissected out from snap-frozen white matter. Brain nuclei were fluorescence-activated nuclear sorted and treated with Transposase Tn5. Gel beads-in-emulsion was generated, barcoded followed by library construction and sequencing.
2024-06-21 | GSE231584 | GEO
Project description:The mammalian lens consists of an aged core of quiescent cells enveloped by a layers of mature fully elongated and younger, continuously elongating and transcriptionaly active cells. In the present study, we compared global gene expression profiles in elongating and mature, non-elongating fiber cells. The microarray-based comparison of gene expression profiles was performed using Agilent 22K Mouse Oligo microarrays with two rounds of RNA amplification from samples obtained by laser capture microdissection (LCM) of paraffin-embedded lens sections. Lenses were removed and immediately fixed using 4% paraformaldehide or methanol-based UMFIX reagent (Sakura Finetek USA, Inc.). To map the exact location of both elongating and maturing fibers on the lens slices, lenses were vibratome-sliced (Vibratome 1000, St. Louis, MO) as described previously and the GFP expression pattern was captured by confocal microscopy. For RNA extraction lens tissue was sliced into 5 micron-thick paraffin sections and microdissected using LCM (Leica Microsystems, Bannockburn, IL). The mid-saggital slices were used both for syncytium border measurements and for LCM. Control measurement (data not shown) confirmed that similar rates of shrinkage in paraformaldehyde- and UMFIX-fixed samples did not affect precision of LCM dissection. Fiber cell samples were dissected out of 5 micron-thick slices. In one experiment we typically processed about 40 slices, which was sufficient to collect the minimum of 200 zone-specific cells pooled from P5 littermate lenses. This sample size provided a reliable representation of RNA species in experimental procedure originally designed and tested for just 1-10 cells. Localization of the syncytium border characterized by the abrupt change of GFP labeling pattern was captured by confocal microscopy as described previously. In brief, GFP fluorescence was visualized using LSM510 instrument (Carl Zeiss, NY) equipped with an argon/krypton laser at 488 nm excitation and a 515-565 nm band pass emission filter. Physical parameters of the zones containing young and maturing fibers were measured in fixed lens slices using the morhphometric software provided by Zeiss. Lenses were fixed in 4% paraformaldehyde/PBS and sectioned with a vibratome. Cells from elongating and maturing fiber regions were dissected out using the Leica DMLA laser capture microscope, (Leica Microsystems, Bannockburn, IL). The cut-out pieces containing captured cells were put directly into tubes containing the lysis buffer supplied in the Absolutely RNA® nanoprep kit (Stratagene, La Jolla, CA). Total RNA from the microdissected tissue sections was extracted and purified using the Absolutely RNA nanoprep kit according to the manufacturer's protocol. Caps briefly placed onto the section without laser activation were used as negative controls. Samples from several age-matched lenses were pooled together to obtain differentiation-specific samples for microarray analysis. Target RNA amplification and labeling with Cy-3 or Cy-5 dyes from CyDye Post Labelling Reactive Dye Pack (Amersham, USA) was carried out in two rounds using the Amino Allyl MessageAmp� aRNA Kit (Ambion, USA) as specified by the manufacturer. Quality and size distribution of the targets were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The Amino Allyl MessageAmp aRNA Kit is configured to incorporate the modified nucleotide, 5-(3-aminoallyl)-UTP (aaUTP) into the aRNA during in vitro transcription. Once purified and fragmented, the dye labeled aRNA was used for microarray hybridization. Labeled amplified RNA was hybridized to the 22K Mouse Oligo microarrays (Agilent Technologies)) according to the manufacturer's instructions. After hybridization microarrays were washed and scanned using GenePix 4000A (Axon Instruments,Inc.).
2008-06-22 | E-GEOD-2083 | biostudies-arrayexpress
Project description:Purpose: to depict the single-cell map and reveal the heterogeneity of hPSC derived macrophages Methods: Differentiation day 14 iMacs were dissociated to obtain a single-cell suspension. scRNA-seq and scATAC-seq library preparation and sequencing were done by CapitalBio Technology (https://www.capitalbiotech.com/). scRNA-seq, the cell suspension was immediately loaded onto the Chromium Single Cell Controller (10× Genomics) for droplet formation. cDNA and subsequent sequencing library were generated using Chromiun Single Cell 3’ Reagent Kit (v3 Chemistry) (10× Genomics) according to the instruction manual. For sc-ATAC-seq, cell nuclei were extracted with Chromiun Nuclei Isolation Kit (v2 Chemistry) (10x Genomics) and then loaded onto the Chromium Single Cell Controller for droplet formation. Results: ScRNA-seq and scATAC-seq data of differentiation day 14 iMacs from hPSCs Conclusions: day 14 differentiation culture, which consisted primarily of monocytes and macrophages.
2024-05-23 | GSE234577 | GEO
Project description:In 10X Genomics Visium Spatial Gene Expression (ST), the resolution for distinguishing neighboring cells can be improved using data integration with single-cell/single-nuclei transcriptomics profiles. Besides, depending on the cell type and tissue, nuclei size may vary significantly to an extent that it may exceed the thickness of tissue slices. This may jeopardize capturing full transcriptomics profile of single slice due to the improper/incomplete incision of nuclei during cryosectioning process and this may cause drawbacks in downstream analysis. To monitor the probable consequences, we monitored the effect of consecutive slices data integration (CSDI) on improvement of cell type clustering and annotation through transferring cell labels from a single-nuclei transcriptomics dataset to ST. To do so, two consecutive slices from the orbitofrontal neocortex and temporal neocortex of two post mortem brain samples were obtained and their spatial transcriptomics profiles were retrieved using 10x Genomics Visium Spatial Gene Expression protocol. Using CSDI, not only the number of identified clusters were increased and the inconsistency between the pattern of clusters in consecutive slices was resolved, but the layered-structure of gray matter was unveiled. Besides, only after CSDI the transferred annotations from single-nuclei transcriptomics to ST could match the microscopic results. CSDI can improve the ST clustering and cell type annotation by providing the full signals coming from all cell types of single slice of tissue. The codes in R programming language are publicly available at https://github.com/ElyasMo/ST_snRNA-seq
2023-05-12 | GSE184510 | GEO
Project description:Purpose: The goal of this study is to determine whether hepatic deletion of Mettl3 affects chromatin accessibility. Methods: Liver samples were pooled from livers of Mettl3 HKO and Mettl3 flox/flox mice (n=4 for each group). Nuclei was extracted from liver samples, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37°C for 30 min. Equimolar Adapter1 and Adatper 2 were added after transposition, PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed with Qubit. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufactuer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated. ATAC-seq analysis was performed using a standard protocol. Results: The hepatic chromatin accessibility in Mettl3 flox/flox and HKO mice were characterized.
2021-10-29 | GSE141245 | GEO