2dcc7cdc-e1d1-4c57-95bf-3713463dec41 - samples
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ABSTRACT:
PROVIDER: EGAD00001006863 | EGA |
REPOSITORIES: EGA
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Project description:HD and control patient-derived induced pluripotent stem cells were used to generate medium spiny neuron-like cells. four control in duplicate and three HD samples in duplicate with CAG repeat length in juvenile onset range were differentiated as biological growth replicates (separate differentiations) into medium spiny neuron-like cells. Cells were treated with LNPs with siRNA for knockdown of PIAS1 or a luciferase control. Total RNA was isolated using the Qiagen RNeasy Kit and QIAshredders for cell lysis. For mouse data WT and HET GFP+ micro-dissected flash frozen brain regions (striatum) were homogenized in TRIzol reagent (Invitrogen) and RNA were collected and purified using RNEasy Mini kit (Qiagen). Mice were treated with AAV2/1 expressing either a miRNA against Pias1 (miPias1.3), a control scrambled miRNA (miSafe). 1 µg of RNA with RIN values >8 were used for library preparation using the strand specific Illumina TruSeq mRNA protocol. Libraries were sequenced on the HiSeq 4000 using 100 cycles to obtain paired-end 100 reads.
2021-01-01 | GSE162349 | GEO
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Project description:The goal of this study is to identify genomic signatures predicitve of cell-of-origin in acute myeloid leukemia 50K bulk leukemia (GFP+) cells from the spleen of recipient mice were sorted directly into 350μl of RLT buffer (Qiagen) and flash-frozen. Total RNA was isolated according to manufacturer’s protocols (Qiagen) including DNase treatment, and quality was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Nano kit. Amplified cDNA was sheared to approximately 300bp using a Covaris E220 Focused Ultrasonicator. RNA-seq library preparation used the TruSeq DNA sample prep kit v2 (Illumina). Libraries were sequenced on the Illumina HiSeq 2000 platform.
2016-07-12 | GSE74690 | GEO
Project description:Determine whole genome RNA sequence information from 2-cell stage mouse embryos (20 embryos per sample). To use Ovation RNA-Seq System V2 kit for cDNA preparation of both mRNA and non-polyadenylated transcripts. Paired-end sequencing.
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Project description:<p><strong>BACKGROUND:</strong> Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at - 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs flash-freezing. To do this stool was collected from an infant's diaper was divided into 2 aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different timepoints to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).</p><p><strong>RESULTS:</strong> Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 timepoints (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given timepoint clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p < 0.05) at timepoint 2 relative to timepoint 1 was similar in flash-frozen vs ambient temperature storage. Changes in microbiota modified metabolites over time were also consistent across both methodologies.</p><p><strong>CONCLUSION:</strong> Ambient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.</p>
2021-01-27 | MTBLS201 | MetaboLights
Project description:Mammary glands of 8 adult (10 week-old) female mice were collected. Freshly sorted basal and luminal epithelial cells were submitted to a Fluidigm C1 System machine for single cell capture and cDNA synthesis. Cells were visualized under the microscope to ensure integrity of the captured single cells prior to cDNA preparation. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina Hiseq 2000 to achieve 100bp paired-end reads.
2017-11-20 | GSE95432 | GEO
Project description:Comparison of TopHat alignments and assessment of spurious splice junctions for 32nt and 76nt read lengths. Total RNA from 2-week-old Arabidopsis thaliana (ecotype Columbia) seedlings grown on MS plates was isolated using RNeasy Plant Mini Kit from Qiagen. To remove any contaminating DNA, RNA was treated with DNAse. Isolation of poly (A) mRNA and preparation of cDNA library were carried out using the Illumina TrueSeq RNA kit. Sequencing (72 cycle) was done on Illumina Genome Analyzer II.
2011-09-24 | GSE32318 | GEO
Project description:Brains were harvested from 2-month-old adult, p12 day old mice and e18.5 day mice embryo. Brain regions of interest (Visual cortex, Thalamus and Hippocampus) were dissected from fresh brains under a light microscope. The tissues were enzymatically dissociated by incubating in 5 ml of a papain solution containing Earle’s balanced salts (EBSS, Sigma, St. Louis, E7510), D(+)-glucose (22.5mM), NaHCO3 (26mM), DNase (125U/ml, Worthington, Lakewood, LS002007), papain (9 U/ml, Worthington, Lakewood, NJ, LS03126), and L-cysteine (1mM, Sigma, St. Louis, C7880) at 35 °C (incubation time varied with developmental stage: 60 minutes for adult and post-natal tissues, 30min for e18.5). The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during papain treatment. Following papain treatment, the tissue was mildly dissociated using the gentleMACS dissociator (Miltenyi Biotec, Germany), followed by gentle trituration using a 5ml pipette. The enzymatic reaction was stopped by adding 5ml of inhibitor buffer (Ovomucoid Trypsin inhibitor, Worthington, Lakewood, LK003182) and centrifuged at 130g for 5 minutes. The cell pellet was then resuspended in 10 ml RPMI1640 medium (Sigma, St.Louis, 11875093) containing 0.02% BSA and 12.5U/ml DNase and strained through a 37um filter (STEMCELL Technologies, BC, 27215) to remove undissociated cell clumps. This cell suspension was then depleted for debris using the Dead cell removal kit (Miltenyi Biotec, Germany, 130-090-101) according to manufacturer’s instruction. Live single cells were enriched by FACS sorting cells (LIVE/DEAD cell viability assay; Molecular Probes, Life Technologies). Single cells were counted and diluted to 250-500 cells per microliter. Cells were loaded, captured on onto C1 integrated fluidic circuits IFC (5- to 10-μm chip) for cell lysis, reverse transcription with oligo (dT) primers and amplification of cDNA on a C1 Single-cell Auto Prep System according to the mRNA-seq protocol of the manufacturer (Fluidigm, USA). After cells were captured on the chip, they were imaged using phase-contrast and fluorescence microscopy before cell lysis. Resulting cDNA was quantified with a Quant-iT PicoGreen dsDNA Assay Kit (PN P11496; Life Technologies), and quality was checked with High Sensitivity DNA Reagents (PN 5067-4626) according to the manufacturer's instructions (Agilent Technologies). Only cells with high-quality cDNA, with a concentration higher than 0.05ng/ul were processed for subsequent library preparation. Library preparation was performed using Nextera XT DNA Sample Preparation Kit (Illumina) as described in the Fluidigm manual. Following library preparation, cells were pooled and sequenced on an Illumina HiSeq 2500 platform as 1x100bp paired-end reads.
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