B713a0bc-f5da-47f6-bc9f-ea514d594db2 - samples
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ABSTRACT:
PROVIDER: EGAD00001006279 | EGA |
REPOSITORIES: EGA
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Project description:Raw sequencing reads from H3K27ac ChIP and input DNA from lymphoblastoid cells of three TET2 mutation carriers and two wild-type family members were quality and adapter trimmed with cutadapt version 1.16 in Trim Galore version 0.3.7 using default parameters. Trimmed reads were aligned to hs37d5 reference genome using Bowtie2 (version 2.1.0). Duplicate reads were removed with samtools rmdup (v1.7). Fragment coverage of paired-end reads was calculated from bam files with BEDtools genomecov (v2.26.0).
| EGAD00010001671 | EGA
Project description:Purpose: The goal of this study is to compare NGS-derived transcriptomes of wild type mouse small interstinal organoids and organoids deficient for the enzyme acetyl-coa-carboxylase (ACC) 1 (Acaca). Methods: mRNA profiles were generated from organoids at 24h and 96h upon in vitro deletion of the Acaca gene and the respective non-deleted wild type controls in triplicates. Total RNA was isolated using RNeasy Micro Kit (Qiagen) and libraries were prepared using NEBNext Single Cell/Low Input RNA Library Prep (New England BioLabs). The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S2 Reagent Kit (100 cycles, paired end run). Each FASTQ file gets a quality report generated by FASTQC tool. Before alignment to reference genome each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using Trim Galore! wrapper tool. Reads shorter than 20 bp were removed from FASTQ file. Trimmed reads were aligned to the reference genome (GRCm38.) using open source short read aligner STAR (https://code.google.com/p/rna-star/) with settings according to log file. Results: the sequencing depth of our libraries is > 3 x107 reads per RNA sample. PCA analysis revealed a close relationship between WT and ACC1-deficient organoids at 24h, whereas samples at 96h were distinct from each other. Lack of ACC1 strongly reduced the expression of genes associated with intestinal epitelial stem cells. Moreover, GSEA revealed downregulation of pathways associated with DNA replication, cell cycle and chromosome segregation in ACC1-deficient organoids. Conclusions: Our study highlights the importance of ACC1-mediated cellular fatty acid synthesis for the maintenance of intestinal epithelial stem cells in mouse intestinal oganoids.
2021-11-10 | GSE188386 | GEO
Project description:Evaluation of modulation of the innate immune response during H1N1 infection. The modulatory effect of Single-stranded oligonucleotides (ssON) on monocyte-derived dendritic cells (MoDCs) are evaluated. RNAseq data are used to study the effect on the transcriptome of MoDCs, during infection with simultaneous addition of ssON. Further mechanistic information are added via RNAseq data on poly I:C stimulated MoDCs (Toll-Like Receptor 3 agonist). Control samples are included to perform differential expression analysis. Provided are the fastq files, obtained in the following manner: The RNA sequencing was performed with the TruSeq RiboZero kit from Illumina, 25 M reads per sample and 2x125bp. Read quality were assessed using FastQC (Version 0.11.5) Trim Galore (Version 0.3.6) was used for adapter removal and quality trimming with a quality threshold of 20 on the Phred scale. Count files was created out of the trimmed fastq by mapping high-quality reads to Homo sapiens UCSC hg38 (GRCh38.77) reference genome using STAR aligner (version 2.5) with default values and the parameter out Reads Unmapped set to Fastx in order to extract the unmapped reads. After STAR alignment, the count data for the aligned reads were generated with HTSeq-count (version 0.6.1). The-m parameter was set to union.
2019-05-03 | E-MTAB-7803 | biostudies-arrayexpress
Project description:Levodopa-induced dyskinesia (LID) is a persistent behavioral sensitization that develops after repeated levodopa (L-DOPA) exposure in Parkinson disease (PD) patients. we used reduced representation bisulfite sequencing to determine the methylation status of cytosines genome wide at base pair resolution following a parkinsonian-like lesion and LID development. Due to the enrichment of RRBS, we focused our analysis to cytosines in a CpG context and observed extensive locus-specific changes in DNA methylation, including a preponderance of demethylation, in the dorsal striatum following the development of dyskinetic behaviors in our animal model system. Changes in DNA methylation were concentrated in putative regulatory regions of many genes known to be aberrantly transcribed following L-DOPA exposure and enriched for genes relevant to mechanisms of synaptic plasticity. In the areas of the genome exhibiting the highest levels of effect, the magnitudes of change to methylation were strongly correlated with dyskinetic behaviors. Rats were given a unilateral dopaminergic lesion to the left medial forebrain bundle with 6-OHDA to destroy at least 90% of TH positive axons. Animals were allowed to recover for 3 weeks and then given daily injections of L-DOPA (6 mg/kg) for seven days to establish stable dyskinesia. Animals were sacked 3 hrs following the final L-DOPA injection and the dorsal striatum was immediately dissected and flash frozen. Striatal tissue samples were processed for nucleic acid isolation using the AllPrep DNA/RNA Mini Kit (Qiagen) following the manufacturer's instructions. One μg of gDNA sample was used for library construction. Bisulfite converted DNA libraries were produced and adaptor ligated, and single-end reads were sequenced on an Illumina HiSeq-2500 following library QC. Bisulfite conversion efficiency was greater that 98% for all of the samples and we obtained an average of 68 million reads per library. Quality control on raw reads was performed with FastQC (version 0.10.1, http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc), and adaptor trimming and removal of trimmed reads shorter than 20 bp was performed with Trim Galore (version 0.3.7, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore). Trimmed reads were mapped to the UCSC Rattus norvegicus rn5 genome with the methylation-aware mapper bismark (version 0.13.0). Samtools (version 0.1.19â??96b5f2294a) was used to sort SAM files produced by bismark and de-duplicate reads. SAM files were analyzed using MethylKit (version 0.9.2) in R (Akalin et al., 2012). Reads were filtered based on coverage, with a cutoff of at least ten reads per site, and normalized for each samples coverage before analysis. The genome was tiled at 250 bp and regions were counted if they contained at least 2 identified CpGs per tile. Differential methylation was defined as a sliding linear model correct p-value of <0.01 and, for highly dynamic regions, included at least a 5% change.
2016-06-16 | E-GEOD-83385 | biostudies-arrayexpress
Project description:Total RNA was purified from TIG-3 cells transfected with siControl or siPRPF19 using miRNeasy Mini Kit (QIAGEN) with RNase-free DNase (QIAGEN). Library preparation, sequencing, and data analysis were performed by DNAFORM (Yokohama, Kanagawa, Japan). Quality and quantity of extracted RNA was assessed by NanoDrop 8000 Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific) and Agilent RNA 6000 Nano kit of BioAnalyzer 2100 System (Agilent Technologies). RNA-seq library was prepared using SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara Bio) following the manufacturer’s instruction. In brief, ribosomal RNA was depleted employed RiboGone which included in the kit. After that, first strand cDNA synthesis was performed using N6 primer. The first-stranded cDNA was purified using equal volume of AMPure XP beads (Beckman Coulter). The purified cDNA was amplified into Illumina-specific RNA-seq libraries by PCR. The libraries were sequenced on a Hiseq sequencer (Illumina) to generate 150 nt paired-end reads. The quality of sequence data was first assessed using FastQC (ver. 0.11.7). Raw sequence reads were then trimmed and quality-filtered with Trim Galore! (ver. 0.4.4), Trimmomatic (ver. 0.36), and cutadapt (ver. 1.16) software. Trimmed reads were mapped to the human reference genome (GRCh38.p10) using STAR (ver. 2.6.1a).
2021-07-20 | GSE168391 | GEO
Project description:The RNA-seq experiment was designed to determine the effects of blastocyst transfer on the transcriptome of whole placentas at embryonic day (E) 10.5. C57Bl/6 blastocysts that were from natural matings without superovulation were transferred into the uteri of pseudopregnant B6D2F1 females (at gestational day 2.5 equivalent). RNA libraries were prepared from whole male placentas at E10.5 (N = 4 placentas) from the transfer group alongside RNA libraries from control placentas from C57Bl/6 conceptuses derived from natural matings and did not undergo the transfer process (N = 4 placentas). Libraries were muliplexed and pooled for sequencing on the Illumina NextSeq500 platform with 75-base-pair single-end reads. Sequencing was performed in duplicate to provide at least 18 million reads per sample, and 1% PhiX Control (Illumina) spike-in was used. Quality control of Fastq files was performed using FastQC and fastq_screen. Sequences were trimmed with Trim Galore!, and alinged to GRCm38 mouse genome using STAR aligner.Alignments were processed using custom ClusterFlow (v0.5dev) pipelines and assessed using MultiQC (0.9.dev0). Gene quantification was determined with HTSeq-Counts (v0.6.1p1). Additional quality control was performed with rRNA and mtRNA counts script, feature counts (v 1.5.0-p2) and qualimap (v2.2). Differential gene expression was performed with DESeq2 package (v1.16.1, R v3.4.0). Read counts were normalised on the estimated size factors.
2019-12-31 | E-MTAB-8036 | biostudies-arrayexpress
Project description:Capture and processing of single skin and blood T cells was performed using the Fluidigm C1 Autoprep system. Cells were loaded at a concentration of 2,000 cells/µL onto C1 integrated fluidic circuit chips for 5–10-µm cells. All C1 capture sites were microscopically inspected to identify the sites that contained only a single cell. Empty sites and those with multiple cells were excluded from further analysis. External RNA Controls Consortium spike-in RNAs served as a control. The SMARTer® Ultra® Low RNA Kit (Clontech) was used for reverse transcription and cDNA pre-amplification. The single-cell cDNA products from each cell were then used to prepare Illumina sequencing libraries and sequenced as paired-end 150-base reads on the Illumina HiSeq 4000 platform. Quality control was performed using FastQC v. 0.11.7 (Babraham Bioinformatics), and the adaptors and low-quality bases with a Phred quality score <20 were trimmed from the ends of the reads using Trim Galore v. 0.4.4 (Babraham Bioinformatics)
2023-12-28 | GSE224449 | GEO
Project description:Purpose: We identified novel methyl reader domain protein in P. falciparum. Using Next Generation Sequencing, we studied the differential gene expression in PfCDP conditionally deleted P. falciparum versus wild type. Methods: Transcriptome analysis of PfCDP KO vs WT P. falciparum using high throughput RNA sequencing in triplicate (Three controls and three KO samples). The total RNA was isolated from P. falciparum WT and PfCDP KO lines, the quality of RNA was assessed using Nanodrop2000, and Bioanalyzer 2100. RNA sequencing libraries were prepared with Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit. The double stranded cDNA was purified using JetSeq Beads and the purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext® Ultra™ II Directional RNA Library Prep protocol. The libraries were sequenced on IlluminaHiSeq 4000 sequencer for 150 bp paired-end chemistry. The data obtained from the sequencing run was de-multiplexed using Bcl2fastq software v2.20 and FastQ files were generated based on the unique dual barcode sequences. The sequencing quality was assessed using the FastQC software ver. 0.11.9. The adaptor sequences and low quality bases (phred quality cutoff set at 30) were trimmed from the reads using the Trim Galore tool ver. 0.6.5. The reads that survived quality check were aligned to the Plasmodium falciparum 3D7 isolate genome (ver. 46) using the Hisat2 read alignment tool. The library strandedness was set as firststrand in accordance with the strand specific library preparation protocol. The alignement output files in the sam format were sorted and converted to the bam format using the Samtools utilities (ver 1.10). Results: The transcript abundance count data was generated using the HTSeq-count function (ver. 0.12.4). Differential gene expression analysis was performed using the DESeq2 package (ver. 3.11) in R programming environment. Genes were classified as deregulated on the basis of log2fold-change cutoff of +1 (for upregulation) and -1 (for downregulation) and p-value cutoff of 0.05. Gene Ontology analysis was performed using the GO tool hosted by the PlasmoDB web database. Data visualisation of the DGE was performed using custom scripts in R programming environment and with DESeq2 package utilities. The DGE analysis has revealed that up regulation of a subsets virulence genes family with significant p values. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2020-09-26 | GSE158585 | GEO
Project description:This dataset contains scRNA-seq fastq files (trimmed for quality and adapters using Trim Galore) for 3739 intestinal plasma cells of known or unknown antigen specificities from in total 12 individuals (4 untreated coeliac disease patients, 3 treated coeliac disease patients, 5 controls). The data was generated using the Smart-seq2 protocol and sequenced on the Illumina NextSeq500 platform with 75 bp paired-end reads.
| EGAD00001006436 | EGA
Project description:DARPP-32 expression was reduced in oestrogen receptor positive luminal B breast cancer cells (T47D) using siRNA and control and DARPP-32 knockdown cells were subject to stimulation with estradiol17β (E2) or inhibition of protein kinase A (PKA) with PKA inhibitor fragment (6-22) amide prior to RNA sequencing. A bioinformatics pipeline of Trim Galore, Kallisto, DESeq2 and Qiagen Ingenuity Pathway Analysis was used for pathway analysis.
2023-10-30 | GSE204836 | GEO