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Gene expression profile in the human heart across hear regions [RNA-Seq]

ABSTRACT: High-througput sequence was demonstrated with NovaSeq 6000 (Illumina). First, extracted RNAs were purified by poly(A) capture. Resultant mRNAs were then fragmented and reverse-transcribed into single-stranded complementary DNAs (cDNAs). Subsequently, cDNAs were double-stranded by a DNA polymerase. During the polymerase reactions, deoxy UTP (dUTP) were mixed in nucleotide materials. Both ends of double-stranded DNA (ds DNA) were ligated to a 13 bp adapter sequence. Next, the ds DNAs were subjected to PCR amplification for the multi-sized DNA library preparation. NovaSeq Control software v1.4.0 analyzed the sequencing runs and tag sequences classified each read in the raw sequencing data. A total of 21 RNA-seq data were used for human data, three samples each of LA, left atrial appendage (LAA), LV, pulmonary vein (PV), RA, RV, and SA. fastp software (version 0.12.4) was used for Read quality control and adapter removal. Reads were aligned using STAR software (version 2.7.0a).

ORGANISM(S): Homo sapiens

PROVIDER: GSE203367 | GEO | 2022/06/22

REPOSITORIES: GEO

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Altered DNA Methylation in Leukocytes with Trisomy 21

Project description:The primary abnormality in Down syndrome (DS), trisomy 21, is well known, but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. NOTE 1: the analysis in Kerkel et al., PLOS Genet, 2010 was carried out after removing probes for genes on the X and Y chromosomes. NOTE 2: these GEO data have been analyzed with this latest version of BeadStudio; the data in the supplementary tables of Kerkel et al., PLOS Genetics, 2010, were from these same Bead chips, but extracted using an earlier version of this software. This re-extraction does not affect the conclusions in Kerkel et al., PLOS Genetics, 2010.

2010-11-18 | GSE25395 | GEO

aca2dd87-573a-481c-a07b-ab3bd8dc3157 - samples

Project description:RNA-Seq was performed on 249 DS-ALL samples. Library preparation was carried out using TruSeq Stranded Total RNA Library Prep Kit. The libraries were sequenced on a NovaSeq platform with read length of 2ÃƒÂ—101.

| EGAD00001009403 | EGA

NimbleGen Rat Gene Expression Microarrays for 2 samples (icariin/DMSO-treated BMSC)

Project description:Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit; 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization. The 2 gene level files were imported into Agilent GeneSpring GX software (version 11.5.1). Genes that have values greater than or equal to lower cut-off: 100.0 in that at least 1 out of 2 samples (“All Targets Value”) were chosen for data analysis.

2022-07-20 | GSE208583 | GEO

RNA Sequencing analyses of control and HuD KO (Elavl4 null) mouse cortical transcriptomes

Project description:Purpose: The goal of this study was to analyse RNA-seq data to determine the effect of deletion of the RNA-binding protein HuD in transcriptiome-wide alternative splicing and polyadenylation in the neocortex of adult HuD KO vs. wild type littermates (controls) Methods: Cortical mRNA profiles of adult HuD KO (Elavl4 -/-) mice and Control mice were generated by RNA sequencing, in triplicate, using Illumina NovaSeq 6000 platform. The quality of raw RNA-sequencing reads was evaluated using FastQC software (version 0.11.5) and adapters were removed using the Cutadapt (version 1.15) and Trimmomatic (version 0.38) software. Alternative splicing was evaluated using rMATS software (version 4.0.2) and BAM files were converted to BedGraph before examining alternative polyadenylation using DaPars software (version 0.9.1) Methods (cont.): RNA-seq data was aligned to the M musculus genome (UCSC browser, mm10) using STAR (version 2.7.3a), and MultiQC (version 1.8) was used to perform a final quality check on STAR alignment files. If alignments were found to be the same read length and have >80% reads mapped to a unique location, the data was considered good quality and alternative splicing and polyadenylation analyses were performed. Sequence reads per sample were aligned to the mouse genome (build mm10). Results: HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of the proximal polyadenylation signal (PAS), resulting in shorter 3’ untranslated regions (3’ UTRs). Conclusions: HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

2021-03-17 | GSE169023 | GEO

MRNA prolfiles of subcutaneous SK-N-BE(2) xenografts treated with the KIF11 inhibitor 4SC-205

Project description:The aim of these study is to investiate the expression profile of neuroblastoma SK-N-BE(2) xenografts treated with one single dose of the KIF11 inhibitor 4SC-205 for 24 h. RNASeq libraries were prepared following the TruSeq® Stranded mRNA LT Sample Prep Kit protocol and sequenced on NovaSeq 6000 (Illumina). RNA-seq reads were mapped against human reference genome (GRCh38) using STAR software version 2.5.3a with ENCODE parameters. Genes were quantified using RSEM version 1.3.0. Tumors treated with 4SC-205 presented increased expression of genes involved in the G2/M checkpoint and E2F targets. Genes of mTORC1 pathway were found to be less expressed on treated tumors otherwise. This study demonstrates that KIF11 inhibition using 4SC-205 halts cell cycle progression of cancerous cells of neuroblastoma xenografts.

2022-02-06 | GSE166984 | GEO

Antagonistic roles of canonical and alternative-RPA in disease-associated tandem CAG repeat instability

Project description:Ongoing CAG expansions in Spinocerebellar ataxia type 1 (SCA1) and Huntington disease (HD) brains exacerbate disease. Expansions involve aberrant repair of mutagenic slipped-DNAs, formed from single-stranded DNAs. Whether singlestranded DNA-binding proteins prevent or facilitate expansions is unknown. We assessed canonical RPA (RPA1-RPA2-RPA3) and Alternative-RPA (RPA1-RPA4- RPA3), containing primate-specific RPA4. RPA is essential for all DNA metabolism. Alt-RPA has undefined functions. Alt-RPA is upregulated 10-fold in HD and SCA1 patient brains. RPA enhances, while Alt-RPA blocks, correct repair of slipped-CAGs. RPA, but not Alt-RPA, efficiently binds and melts slipped-DNAs. RPA enhances, while Alt-RPA blocks, removal of excess slipped-DNAs by FAN1 nuclease. Protein-protein interactomes reveal unique and shared partners of RPA and Alt-RPA, including expansion-driving proteins. RPA-overexpression in SCA1 mice inhibits CAG expansions in brains, rescuing neuron morphology and motor phenotypes. Modulating repeat mutations is one example involving antagonistic Alt-RPA↔RPA interactions, illuminating questions as to which RPA-mediated processes are also modulated by AltRPA.

2023-10-13 | PXD044158 | Pride

Project description:DNA Recombinase Mediated Assembly of Long Adapter Single Stranded Oligonucleotide Probes (LASSO)

| PRJNA693554 | ENA

Altered DNA Methylation in Leukocytes with Trisomy 21

2010-11-18 | E-GEOD-25395 | biostudies-arrayexpress

Genome-wide DNA methylation analyses by the HELP assay

Project description:The HELP tagging assay was performed on purified genomic DNAs. The protocol was modified for NEBNext Multiplex Oligos for Illumina (NEB) from the original protocol4 (http://wasp.einstein.yu.edu/index.php/Protocol:HELP_tagging). Briefly, genomic DNA was digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. AS and AE adapters were prepared by annealing two oligo DNAs separately. The first Illumina AE adapter was ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking genomic DNA sequence. An A-overhang was created, allowing the ligation of the second Illumina AS adapter. This created both AE-insert-AS products and AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we selectively enriched for the AE-insert-AS product. PCR amplification by NEBNext Multiplex Oligos was performed to generate a single-sized amplicons for Illumina sequencing.

2015-09-10 | GSE51680 | GEO

Gene expression profile of human mesenchymal stromal cells (MSCs) exposed to hypoxic and pseudohypoxic preconditioning

Project description:We performed gene expression profiling by RNA sequencing (RNA-Seq) to fully characterize the gene regulation effect induced by exposure of MSCs to two hypoxic preconditioning methods. We determined the gene expression profile of 6 populations of human MSCs isolated from bone marrow and preconditioned for 6h in 2% O2 (hypoxia) or with 40μM Vadadustat (pseudohypoxia), compared to control cells cultured in 21% O2. There were 5 populations isolated from males and 1 population isolated from a female. The mean age of the BM-MSCs donors was 50.5 years and the median was 44 years. RNA-Sequencing was performed using the Illumina platform. For each library an average of 16 - 20 million read pairs were generated. Quality control checks of the sequencing raw data was conducted with FastQC, while adapter-trimming was performed with BBDUK2. The relative expression of transcripts were quantified for each donor using the Salmon program v0.8.1. Fastq files were mapped to the Homo_sapiens.GRCh37 reference genome using HISAT2 software version 2.1.0. The tximport pipeline was used to import transcript abundance datasets for the differential gene expression analysis for three groups: Control vs. Vadadustat, Control vs. Hypoxia and Vadadustat vs. Hypoxia by DESeq2 software version 1.14.1. Our results provide insight into the transcriptomic effects of these two methods of hypoxic preconditioning of MSCs.

2021-08-04 | GSE180371 | GEO

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